Supporting InformationWani et al. 10.1073/pnas.1011665108SI Materials and MethodsCell Culture, Cloning, and Stimulation Conditions. NIH 3T3 fibroblasts.NIH 3T3 cells were cultured in a complete medium of DMEMHigh Glucose (Invitrogen) supplemented with 10% FCS (Invi-trogen) and 1% penicillin and streptomycin (Invitrogen). Cellswere starved for 16 h in serum-free media and stimulated withPDGF-BB (20 ng/mL) at 37 °C for the time points indicated ineach experiment. The zero time point was collected in the absenceof PDGF-BB.Cloning procedures for WT and Cys124Ser myr-Akt2 and transfection ofNIH 3T3 cells.HumanWTmyr-Akt2gene(obtainedthroughAddgene;Plasmid repository no. 9016) in pcDNA3 vector was subcloned intopEF6/V5-His TOPO vector using pEF6/V5-His TOPO TA expres-sion kit (Invitrogen). This construct was further used to createCys124Ser mutation by site-directed mutagenesis (SDM kit, Stra-tagene) according to the manufacturer’s protocol and using thefollowing primers: forward 5′-CATGGACTACAAGTCTGGCT-CCCCCAGTG-3′ and reverse 5′-CACTGGGGGAGCCAGACT-TGTAGTCCATG-3′. These constructs were transfected in NIH3T3 cells using a standard Lipofectamine 2000-based procedure(Invitrogen), and the transfectants were selected with blasticidin (6μg/mL). Similar procedures were followed to generate and transfectin NIH 3T3 cells the Cys297Ser and Cys311Ser myr-Akt2 mutants,and WT myr-Akt1 and Ser122Cys myr-Akt1. Host plasmids andprimers used to generate themutations are summarized in Table S1.C2C12 cells.C2C12 myoblasts were cultured in a complete mediumof DMEM High Glucose (Invitrogen) supplemented with 10%FBS (Invitrogen) and 1%penicillin and streptomycin (Invitrogen).C2C12myoblasts were then induced to differentiate intomyotubesby changing the proliferation medium to DMEMwith 2% FBS for4 d. Myotubes were starved overnight in serum-free DMEM andwere stimulated next day with PDGF (20 ng/mL), insulin (200nM), or TNF-α (10 ng/mL) for 30 min. After stimulation, cellswere lysed with lysis buffer [50 mM Hepes, 150 mM NaCl, 1 mMEDTA, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 25 mMNaF, 10 μM ZnCl2, protease, and phosphatase inhibitor tablets(Roche), pH 7.5] supplemented with catalase (200 U/mL). Theresultant lysates were incubated on ice for 30 min followed bycentrifugation at 10,000 × g for 10 min to obtain cell free extracts.Similar experiments were conducted in the undifferentiatedC2C12 myoblasts. Akt2 was immunoprecipitated, and kinase as-says were performed as described below (Kinase Assays, Kinaseassay using immunoprecipitated Akt proteins).Transfection of 293-T cells and retroviral infection of Akt2 knockout MEFs.293-T cells were cultured in DMEM High Glucose (Invitrogen)media supplemented with 10% FBS (Invitrogen) on 10-cm culturedishes. Transfection was carried out after the cells were approxi-mately 30–40% confluent. Briefly, 18 μL FuGENE-6 was added toantibiotics and serum-free DMEM so as to have a final volume of250 μL after addition of DNA. Six micrograms of DNA (pMIGRconstructs of murine WT myc-Akt2 and Cys124Ser myc-Akt2;Table S1) were added to the tube, and the mixture of FuGENE-6/DNA was allowed to incubate for 20 min at room temperature.Medium from 293-T cells was aspirated, and 5 mL fresh anti-biotics-free DMEM containing 10% FBS was added to the cells.The mixture of DNA/FuGENE-6 was gradually dropped on thecells. Fresh medium (3 to 4 mL) was added to the 293-T cells thefollowing day. After 36 h, viral supernatant was harvested andfiltered through a 0.45-μm syringe filter. For subsequent infection,polybrene (SantaCruz) was added at a concentration of 8 μg/mL tothe filtered supernatant. For infection, Akt2 knockout mouseembryonic fibroblasts (MEFs) were cultured in DMEM High

Glucose (Invitrogen) supplementedwith 10%FBS (Invitrogen) on10-cm culture dishes. Once 40–50% confluent, medium was aspi-rated and replaced with the viral supernatant/polybrene mixtureenough to cover the cells. Four to five h later fresh DMEM wasadded to feed the cells. A second round of infection of the MEFswas carried out using viral supernatant collected 48 h after trans-fection of 293-T. Infection efficiencies were assessed by Westerndetection of Akt2 expression.Conjugating antibodies to protein A-Sepharose beads. Isoform-specificAkt1, Akt2 antibodies (Cell Signaling nos. 2967, 3063), and V5epitope antibody (Invitrogen) were conjugated to protein A-Sepharose beads using standard protocols. Briefly, 50 μL antibodywas incubated with protein A-Sepharose beads (Zymed) ona nutator for 2 h at room temperature. After incubation the beadswere sedimented at 2,000 rpm for 2 min, and the supernatant wasdiscarded. The beads were washed twice with 1 mL sodium boratesolution (0.2 M, pH 9.0) and then incubated with 1 mL freshlyprepared dimethyl pimelimidate solution (5 mg/mL) on a nutatorfor 25 min at room temperature. After incubation, the beads werewashed twice with 1 mL monoethanolamine solution (0.2 M, pH8.0) and twice with 1× PBS. Finally, the beads were resuspendedin 200 μL sterile PBS and stored at 4 °C. Cross-linked slurry (10–20 μL) was typically added to 100 μg cell lysates for immunopre-cipitation in all experiments.Induction of oxidation in NIH 3T3 cells. For exogenous treatment withH2O2, the NIH 3T3 cells were serum starved overnight. Thefollowing day cells were treated with H2O2 (100 μM, 500 μM,and 1,000 μM) for 30 min at 37 °C before PDGF-BB stimulation(20 ng/mL, 10 min). For endogenous increase in H2O2 throughcatalase inhibition, NIH 3T3 cells were treated overnight with 10mM 3-AT (3-amino-1,2,4 triazole; Sigma) in serum-free mediaand stimulated the following day with PDGF-BB (20 ng/mL, 10min). Lysates from both experiments were normalized, and Akt1and Akt2 proteins were immunoprecipitated using cross-linkedAkt1 and Akt2 antibodies. Similar experiments were carried outto assess sensitivity of WT myr-Akt1 and Ser122Cys myr-Akt1proteins to oxidation from lysates of PDGF-stimulated (20 ng/mL, 10 min) and 3-AT (10 mM) treated NIH 3T3 transfectantsusing V5 antibody cross-linked to protein A-Sepharose. The ki-nase activity of immunoprecipitated Akt1 and Akt2 proteins wasfurther assayed as described below under (Kinase Assays, Kinaseassay using immunoprecipitated Akt proteins). Additionally, thelysates from 3-AT–treated cells were probed for expression ofcatalase using anti-catalase antibody (Santa Cruz).

Treatment of NIH 3T3 Cells with Antioxidative Reagents. ConfluentNIH 3T3 cells were subjected to overnight serum-starvation in thepresence of different concentrations of the mitochondrial elec-tron transport chain (ETC) inhibitor rotenone (Sigma; 1–20 μM)and the antioxidant N-acetyl cysteine (Sigma; 5 and 20 mM).After addition of N-acetyl cysteine to the media, pH was read-justed to 7.5 before starving the cells. NOX inhibitor, VAS-2870(5 μM; Enzo Life Sciences) and the antioxidant PEG-catalase(250–1,000 U/mL; Sigma) were added to the serum-starved NIH3T3 cells 2 h before PDGF stimulation (20 ng/mL). Akt2 wasimmunoprecipitated from the normalized lysates and assayed foractivity using GSK-3α/β substrate as described below (KinaseAssays, Kinase assay using immunoprecipitated Akt proteins).

Assessment of PDGF-Induced Oxidation of Cellular Proteome. Relativequantification of ROS in response to PDGF-BB stimulation of NIH 3T3 cells.Subconfluent NIH 3T3 cells cultured in 60-mm dishes were serum

Wani et al. www.pnas.org/cgi/content/short/1011665108 1 of 12

http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1011665108/-/DCSupplemental/pnas.201011665SI.pdf?targetid=nameddest=ST1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1011665108/-/DCSupplemental/pnas.201011665SI.pdf?targetid=nameddest=STXThttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1011665108/-/DCSupplemental/pnas.201011665SI.pdf?targetid=nameddest=STXThttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1011665108/-/DCSupplemental/pnas.201011665SI.pdf?targetid=nameddest=ST1http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1011665108/-/DCSupplemental/pnas.201011665SI.pdf?targetid=nameddest=STXThttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1011665108/-/DCSupplemental/pnas.201011665SI.pdf?targetid=nameddest=STXThttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1011665108/-/DCSupplemental/pnas.201011665SI.pdf?targetid=nameddest=STXThttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1011665108/-/DCSupplemental/pnas.201011665SI.pdf?targetid=nameddest=STXTwww.pnas.org/cgi/content/short/1011665108

starved overnight and assayed for reactive oxygen species (ROS)by imaging at different PDGF-BB (20 ng/mL) stimulation timesusing CM-H2DCFDA (DCF, 100 μM; Invitrogen). DCF wasadded for 10 min and incubated at room temperature beforeimaging for every time point of PDGF stimulation. The cellswere rapidly washed twice with 1× PBS and visualized withArcturus PixCell II laser capture microscope using 20× objective.Both fluorescence and corresponding phase images were col-lected from four random fields per stimulation, and quantifica-tion of the relative fluorescence intensity was done using ImageJ.Affinity capture of DCP-Bio1 labeled proteins and monitoring of Akt2labeling by DCP-Bio1. NIH 3T3 cells that were starved overnightwere stimulated at 37 °C with 20 ng/mL PDGF-BB (Calbiochem)for various time points or incubated for 10 min with 100 μMH2O2 in starvation media or starvation media alone (control).The cells were quenched with 500 μL lysis buffer per 10-cm plate(50 mM Hepes, 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10%glycerol, 1% Triton X-100, 25 mM NaF, and 10 μM ZnCl2)supplemented with 1 mM Na3VO4, PhosStop and cOmpleteProtease Inhibitor Mixture tablets (Roche Diagnostics), DCP-Bio1 (250 μM to 1 mM), and catalase (200 U/mL). The cell ly-sate was then incubated on ice for 1 h, centrifuged, passedthrough a spin column (Bio-gel P6; Bio-Rad) equilibrated withsame lysis buffer, and assayed for protein concentration using theBio-Rad Protein Assay. For Western blot analyses, the lysateswere normalized and incubated overnight at 4 °C with 20–100 μLstreptavidin-agarose conjugate (Invitrogen). In an additionalexperiment, PDGF-stimulated NIH 3T3 cells were lysed afterindicated time points of stimulation using degassed lysis buffer(pH 5.5) to assess contribution of postlysis oxidation. The lysateswere normalized and incubated with streptavidin-agarose con-jugate as mentioned above. The next day the streptavidin-aga-rose beads were washed twice with 1× lysis buffer, and the DCP-Bio1–labeled proteins were eluted by heating the samples for10 min at 100 °C. To ensure that Akt2 pulldown was not due tointeractions with other DCP-Bio1 or endogenously biotinylatedproteins, control experiments were performed with additionalwashes before elution from the streptavidin-agarose beads asfollows: one wash each with 2 M urea, 1 M NaCl, and 10 mMDTT (all reagents were made in 1× lysis buffer) followed by twowashes with 1× lysis buffer. The samples were then separated bySDS/PAGE, transferred to nitrocellulose membrane, and probedwith Akt1, Akt2, and Akt3 isoform-specific antibodies, and thepSer473/4 Akt1/2 antibody (Cell Signaling nos. 2967, 3063, and9271). For the preliminary redox proteomics experiments thatwere aimed at identifying proteins and the hyperactive cysteinesites that are oxidized in response to PDGF, the same procedurewas followed except the experiment was scaled up (starting ma-terial was 20 mg of PDGF-stimulated lysate), and various en-richment strategies were tested, including preenrichment inphosphoproteins (this resulted in the preliminary identification ofPLCγ1) and postenrichment in DCP-Bio1–labeled peptides rel-ative to the streptavidin enrichment step of DCP-Bio1–labeledproteins (this resulted in the identification of Cys124 Akt2 site).FluoReporter assay for DCP-Bio1 incorporation. The FluoReporter as-say kit from Invitrogen was used according to the manufacturer’sinstructions. For determining incorporation of biotin iodoaceta-mide (Biotin-IAA; Sigma) into total cysteine proteome of NIH3T3 cells, the unstimulated cell lysate was first treated with 6 Murea for 15 min at room temperature followed by DTT (1 mM)for 30 min at 37 °C. Biotin-IAA (10 mM) was added, and thesample was incubated on a shaker for 1 h at room temperature inthe dark. For DCP-Bio1–labeled samples obtained as describedabove, unreacted DCP-Bio1 and other small molecules present inthe samples were first removed using a Bio-Gel P6 spin column.The cell lysates thus processed were digested with protease typeXIV from Streptomyces griseus provided in the kit at 37 °Covernight on a shaker. Biocytin (0–80 pmol) was used to generate

a standard curve. Each lysate and biocytin sample (50 μL) wasincubated with 50 μL of Biotective Green reagent in a 96-well platefor 5 min at room temperature in the dark. Fluorescence of thesamples was measured on a Molecular Devices SpectraMax M2efluorescence plate reader using λex = 495 nm and λem = 519 nm.

Kinase Assays. Kinase assay using recombinant active Akt1, Akt2, andAkt3. Kinase assay was performed with recombinant active Akt1,Akt2, and Akt3 proteins (Active Motif) using the nonradioactiveAkt kinase assay kit (Cell Signaling) according to the manu-facturer’s protocol, with the following modifications. Akt1 andAkt2 proteins were oxidized or reduced with increasing concen-trations of H2O2 (25–500 μM) and DTT (10 μM to 1 mM), re-spectively, for 20 min at room temperature. This treatment wasfollowed by incubation with the substrate GSK-3α/β in the pres-ence of 0.4 mM each ATP and MgCl2 for 10 min at room tem-perature. The kinase assay buffer used for these reactions wasmade fresh every time and lacked 1 mM DTT unlike the kinaseassay buffer provided in the kit. At the end of the incubationperiod the reaction mixture was divided into two, and one set wasquenched using 4× SDS sample buffer in presence of reducingagent β-mercaptoethanol, whereas the other set was quenchedwithout the reducing agent. The samples under nonreducingconditions were subjected to immunoblotting with isoform-specific Akt1 and Akt2 antibodies, and the reduced set of re-action was probed for pGSK-3α/β(Ser9/12). The blots werestripped and reprobed for the substrate control using GST an-tibody. In a parallel experiment, recombinant active Akt1, Akt2,and Akt3 proteins were normalized to the same concentration of0.35 μg/μL with Tris buffer (pH 7.5, containing 1 mM DTT).Normalized protein sample (1 μL) was added to either 19 μL ofkinase assay buffer (control for non-H2O2 treatment) or 18 μL ofthe kinase assay buffer plus 1 μL of 5 mM H2O2. The mixturewas incubated for 20 min at room temperature. An aliquot of 3μL mixture consisting of substrate GSK-3α/β, MgCl2 (10 mM),and ATP (10 mM) (1 μL each) was added to each vial for further10 min incubation. The reaction was quenched by adding 7 μL of4× SDS sample buffer (plus β-mercaptoethanol) for SDS/PAGEand Western blot analysis. Blots were probed for p-GSK-3α/B(Ser9/12) and pAktT308/9 and reprobed for GST.Kinase assay using immunoprecipitated Akt proteins. After Akt1 andAkt2 immunoprecipitation the beads were washed once with 1×lysis buffer and twice with freshly prepared 1× kinase bufferlacking DTT, and kinase assay was performed according to themanufacturer’s protocol with GSK-3α/β (1 μg, GST-linked) sub-strate in presence of 0.4 mM each ATP and MgCl2 at 30 °C for30 min. For experiments that involved comparison of reactionkinetics under native and reducing conditions, tris(2-carbox-yethyl)phosphine (TCEP; 1 mM) was added to the reactionmixture before addition of GSK-3α/β substrate. The resultantreaction mixture was separated on SDS/PAGE gels, transferredto nitrocellulose membrane, and probed for pGSK-3α/β(Ser9/12),pAktT308/9, GST, and Akt1 and Akt2 proteins (Cell Signalingantibody nos. 9327, 9275, 2625, 2967, and 3063).Recovery of Akt2 activity by TCEP reduction after oxidation usingrecombinant purified Akt2 and Akt2 immunoprecipitated from fibroblasts.Kinase activity assay was performed with recombinant active Akt2protein (Active Motif) or with Akt2 immunoprecipitated fromtransfected NIH 3T3 fibroblasts pretreated with catalase inhibitor3-AT using the Akt kinase assay kit (Cell Signaling) as describedabove. The active Akt2 protein was oxidized or reduced with H2O2(250 μM) and TCEP (1 mM, pH 7.0), respectively, for 20 min atroom temperature. This treatment was followed by incubationwith the substrate GSK-3α/β in the presence of 0.4 mM each ATPand MgCl2 for 10 min at room temperature. To another set ofAkt2 reaction, after treatment with H2O2 (250 μM) for 20 min,TCEP (1 mM, pH 7.0) was added and incubated further for10 min at room temperature. Endogenous Akt2 from untransfected

Wani et al. www.pnas.org/cgi/content/short/1011665108 2 of 12

www.pnas.org/cgi/content/short/1011665108

and WT myr-Akt2 from transfected NIH 3T3 cells was im-munoprecipitated using cross-linked Akt2 and V5 antibodies,respectively. TCEP (1 mM, pH 7.0) was added to the two Akt2kinases after washes with kinase assay buffer and incubated for10 min at room temperature just before addition of the substrate.At the end of the incubation period the reactions were termi-nated using 4× SDS sample buffer in presence of reducing agentβ-mercaptoethanol. The resultant reaction mixture was separatedon an SDS/PAGE gel, transferred to nitrocellulose membrane,and probed for pGSK-3α/β (Ser9/12), pAktT308/9, and GSTproteins (Cell Signaling antibody nos. 9327, 9275, and 2625).Assessment of oxidation sensitivity of WT and Cys124Ser myr-Akt2. Hu-man WT and Cys124Ser myr-Akt2 were transiently transfected inNIH 3T3 cells using a standard Lipofectamine 2000-based pro-cedure (Invitrogen), and the transfectants were selected withblasticidin (6 μg/mL). The myr-Akt2 transfected cells werestimulated with PDGF-BB (20 ng/mL, 10 min), and the lysateswere used to pull down WT and Cys124Ser myr-Akt2 proteinswith V5 antibody conjugated to protein A-Sepharose beads. Theendogenous Akt2 was immunoprecipitated from control, un-transfected cells using Akt2 isoform-specific antibody. The im-munoprecipitated WT and Cys124Ser myr-Akt2, along withendogenous Akt2, were subjected to in vitro oxidation with 100μM H2O2 for 20 min on a shaker at room temperature and as-sayed for kinase activity as described above. Similar procedureswere followed to assess the sensitivity to oxidation of Cys297Serand Cys311Ser myr-Akt2 mutants.

MS Experiments. Labeling of oxidized cysteines in Akt2 with dimedoneand identification of labeled cysteines by MS. Recombinant activeAkt2 (21 μM, supplied in Tris buffer containing 1 mM DTT) wastreated with H2O2 (1 mM) in the presence of dimedone (8 mM).The reaction was incubated at room temperature on a shaker for1 h and terminated with 4× SDS sample buffer. The reactionmixture was subjected to SDS/PAGE, and the gel was stainedwith Gel Code Blue (Pierce). The protein bands were excised,and in-gel trypsin digestion was performed according to standardprotocols. The resulting tryptic peptides were further separatedand analyzed by nano-LC (Dionex Ultimate3000 System) cou-pled to a Thermo electrospray ionization linear trap quadrupole(ESI LTQ) mass spectrometer. A typical gradient was run for60 min from 0 to 100% solvent B (80% ACN, 20% H2O, and0.1% formic acid). Solvent A consisted of 5% ACN, 95% H2O,and 0.1% formic acid. The flow rate was set at 200 nL/min ona PepMap 100, C18, 3 μm, 100 Å, 75-μm i.d. × 15-cm column(Dionex; catalog no. 160321). The ESI LTQ mass spectrometerwas operated in selected ion monitoring (SIM) mode for pre-cursor ions corresponding to the peptides containing dimedone-labeled cysteine. The peptide identification was then performedautomatically with the Bioworks 3.3 software.Dimedone labeling of Akt1 and Akt3. Six microliters of recombinantactive Akt1 or Akt3 (0.28 μg/μL in Tris buffer containing 1 mMDTT, pH 7.5) was further diluted by adding 12 μL of 50 mM Bis-Tris (pH 7.5), to which 1.6 μL of 0.1 M dimedone in DMSO wasadded, followed by addition of 0.6 μL of 20 mM H2O2. The la-beling reaction was carried out at room temperature for l h withmixing. After labeling, Akt1 and Akt3 was in-gel digested withtrypsin, and the extracted peptides were analyzed in data de-pendent or SIM mode by nano-LC-MS as decribed above.Oxidation of active Akt2 for the identification of Cys124-Cys297 andCys124-Cys311 intramolecular disulfides by MS. Recombinant activeAkt2 stock (1.6 μL) (1.5 μg/μL in Tris buffer containing 1 mMDTT) was diluted in 7.4 μL of 50 mM Tris buffer (pH 7.5), and1 μL of 10 mM H2O2 was added to oxidize Akt2. After 1-h in-cubation at room temperature, the free thiols in Akt2 were al-kylated by adding 1 μL of 0.5 M freshly made iodoacetamide(IAA) in 0.1 M ammonium bicarbonate (1 h, room temperature,

in the dark). The excess H2O2 and IAA were then removedthrough SDS/PAGE, and the gel was washed by ddH2O threetimes before being stained with Gel Code. The stained bandcontaining the oxidized Akt2 was excised, in-gel digested withtrypsin, and analyzed by nano-LC-MS. The identification of theCys124-Cys297 and Cys124-Cys311 disulfide cross-linked pep-tides was performed both through manual inspection of the MS/MS data, MassMatrix, and using the DBond search softwarev.2.04 available at http://prix.uos.ac.kr/ (1, 2).Disulfide mapping in Akt1 and Akt3 and carbamidomethylation of Cys119in Akt3.Recombinant active Akt1 (0.35 μg/μL) and Akt3 (1 μg/μL)were first normalized to the same concentration of 0.28 μg/μLwithTris buffer (pH 7.5, containing 1 mM DTT) to a total volume of18 μL. Then the Akt1 and Akt3 proteins were further diluted byadding 15 μL Tris buffer (pH 7.5). Fifteen microliters of this finalmix corresponding to ≈2.3 μg Akt1 or Akt3 protein was used forexperiments below. Akt1 or Akt3 proteins were first oxidized byadding 0.7 μL of 10 mM H2O2 for 1-h incubation at room tem-perature. Then 0.8 μL of 1 M IAA (freshly made in 0.1 M am-monium biocarbonate) was added for further 1-h incubation inthe dark to block the free thiols. The Akt1 and Akt3 samples weresubjected to SDS/PAGE, in-gel trypsin digestion, and nano-LC-MS analysis (data-dependent mode) as described above.Determination of postlysis oxidation of Akt2 kinase. Akt2 knockoutcells were cultured to approximately 70% confluency in a six-wellplate and starved overnight in a serum-free medium for the nextday treatment. The starved cells were stimulated by PDGF (20 ng/mL) at 37 °C for 20 min or incubated in starving medium onlyat the same conditions. A Bio-Gel column was equilibratedwith lysis buffer before loading 35 μL of recombinant pure Akt2protein stock (0.76 μg/μL, supplied in Tris buffer containing1 mM DTT) to have a DTT-free Akt2 protein. Three μL of theAkt2 protein was added to the 1 mL lysis buffer right before use.Cells were lysed with the lysis buffer spiked with Akt2 protein inthe presence of 200 μM DCP-Bio1, and the lysate was left on icefor 1 h. Lysis buffer containing the Akt2 protein with or withoutthe DCP-Bio1 was also included as control. After incubation, celldebris was removed, and 15 μL of the supernatant was mixedwith equal volume of 4× SDS sample buffer for SDS/PAGE andWestern blot analysis. The membrane was probed for strepta-vidin-conjugated HRP. In another experiment, the SDS/PAGEgel was stained by Gel Code Blue to visualize proteins. For theimmunoprecipitation experiment, 40 μL of the lysate was in-cubated with 10-μL agarose beads conjugated with anti-Akt2antibody for overnight pulldown at 4 °C. In the following day, thebeads were washed by PBS and boiled in 40 μL of 4× SDSsample buffer to elute the proteins. The supernatant from thebeads was then subjected to the SDS/PAGE and Western blottreatment. Biotinylated AhpC protein was included as positivecontrol for the probe using streptavidin-conjugated HRP. Thesame membrane was stripped and reprobed for Akt2.

Glucose Uptake.Akt2 knockout MEFs transfected/transduced withWT and Cys124Ser myc-Akt2 were serum starved overnight in thepresence or absence of 3-AT (10 mM). Next day, the cells werestimulated with PDGF (20 ng/mL) or insulin (200 nM) for 30 minalong with the unstimulated cells as control. Medium was thenremoved, and cells were washed two times with 1× PBS at roomtemperature. The assay was initiated by the addition of 0.1 mM2-deoxyglucose and 0.5 μCi/mL 2-deoxy-D-[3H] glucose (Perkin-Elmer Life Sciences) and terminated after 10 min by washingcells two times in ice-cold 1× PBS and quenching with 0.05 MNaOH. Uptake of 2-deoxy-D-[3H] glucose was detected inScintiverse BD scintillation mixture (Fisher Scientific) usinga Beckman LS6000SC scintillation counter and was normalizedby protein concentration.

Wani et al. www.pnas.org/cgi/content/short/1011665108 3 of 12

http://prix.uos.ac.kr/www.pnas.org/cgi/content/short/1011665108

1. Choi S, et al. (2010) New algorithm for the identification of intact disulfide linkagesbased on fragmentation characteristics in tandem mass spectra. J Proteome Res 9:626–635.

2. Xu H, Zhang L, Freitas MA (2008) Identification and characterization of disulfide bondsin proteins and peptides from tandemMS data by use of the MassMatrix MS/MS searchengine. J Proteome Res 7:138–144.

PDGF DCP-Bio1Biotin-IAA

--+

-+-

++-

Bio

tin c

onte

nt

(pm

oles

/µg

cell

lysa

te)

320

300

280

20

10

0

FluoReporter biotin assay

IYYSEETSSDQGNEDEEEPKPDGF- +

PLC 1

m/z

A.

B.

Fig. S1. Labeling of sulfenic acid-containing proteins in PDGF-stimulated NIH 3T3 cells. (A) DCP-Bio1 incorporation before and after 20 min PDGF-BB treatmentwas compared with incorporation of biotin iodoacetamide (Biotin-IAA) into the total cysteine proteome. Endogenous biotinylation (-DCP-Bio1) was determinedto be 0.12 ± 0.03 pmol biotin/μg cell lysate (0.6% of the signal observed for DCP-Bio1). (B) MS/MS spectrum of PLCγ1 identified from the streptavidin-agarosepulldown samples of PDGF-stimulated cell lysates that were labeled by DCP-Bio1 (Xcorr 5.4, z +2). Validation by Western blot is included in Inset.

N-acetyl cysteine (20 mM)

PDGF stimulation, min

5 20 5 20

- 250 1000

PEG-catalase, (U/ml)

- - + +

20 20 20

pAkt(T309)

pGSK-3

pGSK-3 (anti-GST)

VAS2870 (5 μM) - +

Rotenone, μM

- 1 10 20

PDGF stimulation, 20 min

pAkt(T309)

pGSK-3

pGSK-3 (anti-GST)

A.

B.

Fig. S2. Source of oxidation of Akt2 in NIH 3T3 cells. (A) Akt2 activity assayed from serum-starved NIH 3T3 cells stimulated with PDGF for 5 and 20 min andpretreated with various antioxidants such as N-acetyl cysteine (20 mM) and PEG-catalase (250 and 1,000 U/mL). (B) Akt2 activity assayed from serum-starved NIH3T3 cells stimulated with PDGF for 20 min and pretreated with NOX inhibitor VAS-2870 (5 μM) and mitochondrial ETC inhibitor rotenone for the indicatedconcentrations. Akt2 activity increased in response to all of the antioxidative treatments relative to the untreated control.

Wani et al. www.pnas.org/cgi/content/short/1011665108 4 of 12

www.pnas.org/cgi/content/short/1011665108

A.

Akt2

PDGF stimulation, min

0 0.5 1 2.5 10 20

pAkt2 (S474 )

(pH 5.5 degassed lysis buffer)Affinity capture: streptavidin-agarose

B.

--

-- +

-+

+ ++ +

-+ + + + + - DCP-Bio1

PDGFAkt2

Lysis buffer

Ctrl: AhpC pre-labeled with DCP-Bio1 (20 kDa)

Akt275

50

37

75503725

Ctrl

SD

S-P

AG

E

WB: Akt2

WB:streptavidin-HRP

MWmarker

Fig. S3. Postlysis oxidation of Akt2 determined in NIH 3T3 and Akt2 KO MEFs. (A) Serum-starved NIH 3T3 cells were stimulated with PDGF (20 ng/mL) for theindicated time points and lysed using degassed lysis buffer (pH 5.5) containing the labeling reagent DCP-Bio1 (pH 5.5) to address postlysis oxidation. Thenormalized cell lysates were used for affinity capture using streptavidin–agarose conjugate and probed for Akt2 and pS474 Akt2 to assess oxidation andphosphorylation status of Akt2, respectively. (B) Top: Akt2 knockout MEFs were stimulated with or without PDGF for 20 min at 37 °C and lysed without re-combinant Akt2 (first two lanes) or with Akt2 (lanes 3 and 4) added into the lysis buffer in the presence of DCP-Bio1. Lysis buffer with Akt2 in the presence(lane 5) or absence (lane 6) of DCP-Bio1 was also tested along with the cell lysate samples. These samples were subjected to SDS/PAGE, and the gel was stainedby Gel Code Blue to visualize the separated proteins. Arrow indicates the spiked Akt2 protein.Middle: Akt2 immunoprecipitation was carried out on samples atTop and probed for Akt2 as a control after stripping of the previous streptavidin signal shown at Bottom. Bottom: Akt2 immunoprecipitation was carried outon samples at Top and probed for streptavidin. Biotinylated AhpC protein was included as a positive control in the last lane.

Wani et al. www.pnas.org/cgi/content/short/1011665108 5 of 12

www.pnas.org/cgi/content/short/1011665108

Akt1 Cys296-Cys310

Akt1 Cys296-Cys310pT308

Akt3 Cys293-Cys307

Akt3 Cys293-Cys307pT305

A.

B.

C.

D.

Fig. S4. (Continued)

Wani et al. www.pnas.org/cgi/content/short/1011665108 6 of 12

www.pnas.org/cgi/content/short/1011665108

E.

F.

Akt1

Akt3

Fig. S4. Positive ion MS/MS spectra of disulfide cross-linked peptides of oxidized Akt1 and Akt3. (A and B) Disulfide cross-linked peptides from Akt1:ITDFGLC296K and TFC310GTPEYLAPEVLEDNDYGR; ITDFGLC296K and pTFC310GTPEYLAPEVLEDNDYGR. (C and D) Disulfide cross-linked peptides from Akt3:ITDFGLC293K and TFC307GTPEYLAPEVLEDNDYGR; ITDFGLC293K and pTFC307GTPEYLAPEVLEDNDYGR. Notations are the same as in Fig. S5D. (E) Positive ionMS/MS spectrum of a triply charged peptide of Akt1 indicating that cysteine 296 (C296) was labeled by dimedone after H2O2 treatment (score 17, identifiedthrough MassMatrix). (F) Positive ion MS/MS spectrum of a triply charged peptide of Akt3 showing that cysteine 119 (C119) was labeled by IAA after Akt3oxidation by H2O2 (XCorr 6.0 identified through Sequest).

Wani et al. www.pnas.org/cgi/content/short/1011665108 7 of 12

http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1011665108/-/DCSupplemental/pnas.201011665SI.pdf?targetid=nameddest=SF5www.pnas.org/cgi/content/short/1011665108

B.

A.

pAkt (T309)

H2O2TCEP

pGSK-3

GSK-3 (anti-GST)

-

-+ +

++

D.

Akt1 Akt2

GSK-3 (anti-GST)

0 50 100 DTT. 0 50 100 DTTH2O2, µM

pGSK-3

Aktmonomer

dimer

3-AT- + - +

wt myr-Akt1 S122C myr-Akt1

100 92.7 87.5 97.4

pAkt (T308)

pGSK-3

GSK-3 (anti-GST)

C.

Fig. S5. MS and mutagenesis experiments confirm Cys124-Cys297 and Cys124-Cys311 disulfides as key posttranslational events leading to inhibition of Akt2activity by oxidation. (A) Kinase assay was performed under nonreducing and reducing conditions with Akt1 and Akt2 proteins, separated by nonreducingSDS/PAGE, and probed for pGSK-3α/β, GST (GSK-3α/β control), and for Akt1 and Akt2 to determine potential dimerization induced by in vitro oxidation. Thetwo arrows on the Akt blots indicate molecular size for Akt monomer and dimer species. (B) Recovery of Akt2 activity by TCEP reduction after oxidation. Therecombinant active Akt2 protein was reduced with TCEP (1 mM, pH 7.0) after oxidation with H2O2 (250 μM) for 20 min at room temperature. (C) Effect ofSer122Cys mutation on Akt1 activity in response to ROS accumulated by 3-AT treatment in PDGF-stimulated NIH 3T3 cells transfected with WT and Ser122CysAkt1. Ser122Cys mutation does not seem to decrease Akt1 activity in response to PDGF and 3-AT treatment. (D) Positive ion MS/MS spectra of disulfide cross-linked peptides of oxidized Akt2 identified using DBond v. 2.04. Upper: Disulfide cross-linked peptide: C124GSPSDSSTTEEMEVAVSK and ITDFGLC297K. Lower:Disulfide cross-linked peptide: C124GSPSDSSTTEEMEVAVSK and TFC311GTPEYLAPEVLEDNDYGR. Notations for fragment ions are as follows: P*, one strand(Upper) of a dipeptide; p*, the other strand (Lower) of a dipeptide; capital letters, fragmentions from peptide P*; small letters, fragment ions from peptidep*; C-34, dehydroalanine ion; C+32, persulfide ion (formed by S-S or C-S bond cleavage reactions), ^ denotes phosphorylation site. DBond search parameterswere precursor mass tolerance 2 Da, fragment ion mass tolerance 0.8 Da, and variable modifications: oxidation(M), phospho(ST), and carbamidomethyl(C).

Wani et al. www.pnas.org/cgi/content/short/1011665108 8 of 12

www.pnas.org/cgi/content/short/1011665108

endogenous Akt2 wt myr-Akt2

- + +- H2O2, 100 μM

pAkt (T309)

pGSK-3

Akt2

B.

C.

pGSK-3 (anti-GST)

- + - +

pGSK-3 (anti-GST)

pGSK-3

pAkt (T309)

H2O2

Wt Akt2 C124S Akt2 A.

IP: Myr-Akt2 IP: Wt Akt2

-- - +

++ -- - +

++ 3-ATTCEP

pGSK-3

pAkt (T309)

Akt2

pGSK-3 (anti-GST)

Fig. S6. Control kinase assay showing inhibition by H2O2 of endogenous Akt2 and transfected WT myr-Akt2 in NIH 3T3 cells. (A) WT myr-Akt2 and Cys124Sermyr-Akt2 proteins immunoprecipitated from lysates of PDGF-stimulated (20 ng/mL, 10 min) NIH 3T3 transfectants were oxidized in vitro with 100 μM H2O2(20 min) before kinase activity assays (one of the biological replicates for Fig. 3D in main text). (B) Akt2 kinase assay performed with endogenous Akt2(untransfected) and with exogenous WT myr-Akt2 (transfected) immunoprecipitated from PDGF-stimulated NIH 3T3 lysates using cross-linked Akt2 and V5antibodies, respectively. H2O2 (100 μM, 20 min) was added to oxidize Akt2 before the assays. (C) Akt2 activity recovery experiment performed with bothendogenous Akt2 (untransfected) and WT myr-Akt2 (transfected) immunoprecipitated from 3-AT treated and PDGF-stimulated NIH 3T3 lysates. Phosphory-lation of GSK-3α/β substrate was monitored as a measure of kinase activity.

A.

catalase3-AT - + - +

- PDGF + PDGF

--

+-

DCP-Bio1 incorporation after catalase inhibition

-+

++

PDGF3-AT

Bio

tin c

onte

nt

(pm

oles

/μg

cell

lysa

te)

B.

30

20

10

0

Fig. S7. Inhibition of catalase by 3-AT enhances the level of H2O2 and sulfenic acid oxidized proteins in NIH 3T3 cells stimulated with PDGF. (A) Western blotanalysis of intracellular catalase after overnight treatment with 3-AT (10 mM). (B) Pretreatment with catalase inhibitor 3-AT (10 mM) increases the in-corporation of DCP-Bio1 in NIH 3T3 cells as a result of higher levels of intracellular H2O2.

Wani et al. www.pnas.org/cgi/content/short/1011665108 9 of 12

www.pnas.org/cgi/content/short/1011665108

0 100

500

1000

pAkt (S473/4)

-actin

H2O2 ( M)

NIH-3T3 cell lysate

pAkt (T308/9)

-actin

A. B.

C.

pAkt (S474)

pAkt (T309)

0

100

H2O2 ( M)

E.

pAkt (S474)

pAkt (T309)

3-AT (10 mM)- + - +

Endogenous Akt2 IP

PDGF

PD

GF

Insu

lin

TN

F-

pAkt (S474)

pAkt (T309)

C2C12 cells, IP:Akt2

F.

D.

Wt myr Akt2 Akt2 IP

pAkt (S473/4)

VAS-2870 (5 M)

3-AT (10 mM)

- - - + - - -

- + - - - - -

- - - - 1 10 20

PDGF

-actin

pAkt (T308/9)

-actin

NIH-3T3 cell lysates

Rotenone ( M)

PEG-catalase ( M))

NAC (20 mM)

Rotenone (10 M)

- - - 250 1000 -

- + - - - -

- - - - - +

pAkt (S474)

pAkt (T309)

PDGF

NIH-3T3 cells, IP:Akt2

Fig. S8. Comparable phosphorylation at the two activation sites of Akt2 kinase. (A) NIH 3T3 cell lysates from Fig. 2D (main text) were probed for pAkt (T308/9)and pAkt (S473/4). (B) Lysates from Fig. 2E (main text) and Fig. S2B were probed for pAkt (T308/9) and pAkt (S473/4). (C) Akt2 immunoprecipitated from NAC-,PEG-catalase-, and rotenone-treated NIH 3T3 lysates (from Fig. S2) probed for pAkt (T309) and pAkt (S474). (D) Recombinant purified Akt2 kinase was treatedwith 100 μM H2O2 and probed for pAkt (T309) and pAkt (S474). (E) Endogenous Akt2 and transfected WT myr-Akt2 immunoprecipitated from 3-AT–treatedlysates of NIH 3T3 cells (Fig. S6A) were also probed for pAkt (T309) and pAkt (S474). (F) Akt2 immunoprecipitated from PDGF-, insulin-, and TNF-α–treated(30 min) C2C12 myotubes (Fig. S9A) were probed for pAkt (T309) and pAkt (S474).

Wani et al. www.pnas.org/cgi/content/short/1011665108 10 of 12

http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1011665108/-/DCSupplemental/pnas.201011665SI.pdf?targetid=nameddest=SF2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1011665108/-/DCSupplemental/pnas.201011665SI.pdf?targetid=nameddest=SF2http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1011665108/-/DCSupplemental/pnas.201011665SI.pdf?targetid=nameddest=SF6http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1011665108/-/DCSupplemental/pnas.201011665SI.pdf?targetid=nameddest=SF9www.pnas.org/cgi/content/short/1011665108

B.A.

C.

PDGF Insulin

TCEP

IP: Akt2

α

- - + +- +73 99.8 64.5 99.7 87.2 99.8

0 5 15 30 60

α stimulation, min

Affinity capture: Captavidin agarose

pAkt(T309)

pGSK-3

GSK-3 (anti-GST)

Akt2

C2C12 myotubes

Untransfected control Wt-myc-Akt2 C124S-myc-Akt2

- + - + - + - + - + - +

- - + + - - + + - - + +

GSK-3 (anti-GST)

C2C12 myoblasts

pAkt(T309)

pGSK-3

PDGF Insulin

- + - + - +

Control

IP: Akt2

PDGF, min Insulin, min

0 5 30 5 30

Affinity capture: Captavidin agaroseAkt2

TCEP

D.DCP-Bio1 pY

Rel

ativ

e m

ean

inte

nsity

PDGF stimulation time, min

0 5 10 15 20 300.0

0.5

1.0

1.5

2.0

2.5ROS

PDGF stimulation time, min0 5 10 20 30

Rel

ativ

e m

ean

inte

nsity

0

5

10

15

20

Fluo

resc

ence

PDGF stimulation time (min)

Pha

se

0 5 10 20 30

pAS160

pFOXO1/3aThr24/32

pGSK-3 Ser21/9

3-AT (10 mM)

PDGF

Akt2

-actin

Fig. S9. Effect of PDGF, insulin, and TNF-α on oxidation and activity of Akt2 in C2C12 myoblasts vs. myotubes and the effect of WT and C124S Akt2 onsignaling in Akt2 knockout MEFs. (A) Differentiated C2C12 myotubes were stimulated with PDGF, insulin, and TNF-α for 30 min followed by Akt2 pulldownfrom the normalized lysates for assaying activity using GSK-3α/β substrate in the presence and absence of incubation with TCEP (1 mM, pH 7.5). Akt2 activityincreased significantly with TCEP treatment after PDGF and TNF-α treatment in the myotubes relative to insulin. Lower: DCP-Bio1–labeled lysates from C2C12myotubes over a time course of TNF-α stimulation were captured with captavidin–agarose conjugate and probed for Akt2. TNF-α–dependent increase in DCP-Bio1 labeling was noted in the myotubes. (B) C2C12 myoblasts were stimulated with PDGF and Insulin for 30 min along with the unstimulated control, and Akt2was immunoprecipitated for assaying activity using GSK-3α/β substrate in the presence and absence of incubation with TCEP (1 mM, pH 7.5). Akt2 activityincreased significantly with TCEP treatment after PDGF and insulin treatment in the myoblasts. Lower: DCP-Bio1–labeled lysates from C2C12 myoblasts ob-tained after 5 and 30 min stimulation with PDGF and insulin were captured with captavidin–agarose conjugate and probed for Akt2. Both PDGF and insulininduced Akt2 oxidation in the myoblasts. (C) Lysates from 3-AT treated (10 mM), uninfected, and WT and C124S myc-Akt2 infected Akt2 knockout MEFs were

Legend continued on following page

Wani et al. www.pnas.org/cgi/content/short/1011665108 11 of 12

www.pnas.org/cgi/content/short/1011665108

probed for phosphorylation of AS160, FOXO1/3a, and GSK-3 α/β. (D) Time course of PDGF-induced oxidation and phosphorylation in NIH 3T3 cells. (Left) ROSwas measured by DCF imaging over the indicated time course of PDGF stimulation, and the mean fluorescence intensity was quantified from four fields perstimulation time using ImageJ. Corresponding P values for 5 min: 0.0290; 10 min: 0.012; 20 min: 0.001; and 30 min: 0.013. (Right) DCP-Bio1 incorporation intocellular proteins and phosphorylation of cellular proteins was detected by immunoblotting using anti-biotin and 4G10 anti-phosphotyrosine antibodies, re-spectively. Shown here are means and SDs for minimum of three replicates in each case. (Lower) A phase image and its corresponding fluorescence image pertime point of PDGF stimulation for one of the four fields is depicted for visual reference.

Table S1. Constructs and primers

Construct Tag(s) Primers Experiment

pEF6/TOPO construct ofmurine Akt1 and murineSer122Cys Akt1

Myr, tag at N terminus andV5, His tag at C terminus

For Ser122Cys mutation:F: 5′-GAGATGGACTTCCGGTGTGGC

TCACCCAGTGACAAC-3′R: 5′-GTTGTCACTGGGTGAGCCACA

CCGGAAGTCCATCTCC-3′

Akt1 activity assay in NIH 3T3 fibroblaststransfected with these constructs

pMIGR retroviralconstruct of murine Akt2and murine Cys124SerAkt2

Myc tag at N terminus (1) For Cys124Ser mutation:F: 5′-CATGGATTACAAGAGCGGCTC

CCCCAGTGAC-3′R: 5′-GTCACTGGGGGAGCCGCTCTT

GTAATCCATGG-3′

Glucose uptake assay and Akt2 acitivty inAkt2 knockout MEFs transfected withthese retroviral constructs

pEF6/TOPO construct ofAkt2 and Cys297Ser Akt2

Myr, HA tags at N terminusand V5,His tag at C terminus

For Cys297Ser mutation:F: 5′-CTGACTTTGGCCTCAGCAAAG

AGGGCATC-3′R: 5′-GATGCCCTCTTTGCTGAGGCCA

AAGTCAG-3′

Akt2 activity assay (with H2O2 after IPfrom NIH 3T3 fibroblasts transfectedwith these constructs)

PEF6/TOPO construct ofAkt2 and Cys311Ser Akt2

Myr, HA tags atN terminus andV5, His tag at C terminus

For Cys311Ser mutation:F: 5′-CATGAAAACCTTCTCTGGGACC

CCGGAG-3′R: 5′-CTCCGGGGTCCCAGAGAAGGT

TTTCATG-3′

Akt2 activity assay (with H2O2 after IPfrom NIH 3T3 fibroblasts transfectedwith these constructs)

1. Zhou GL, et al. (2006) Opposing roles for Akt1 and Akt2 in Rac/Pak signaling and cell migration. J Biol Chem 281:36443–36453.

Wani et al. www.pnas.org/cgi/content/short/1011665108 12 of 12

www.pnas.org/cgi/content/short/1011665108