supplementary information · supplementary information preso1 dynamically regulates group i...
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Supplementary Information
Preso1 dynamically regulates group I metabotropic glutamate receptors.
Jia–Hua Hu, Linlin Yang, Paul J. Kammermeier, Chester G. Moore, Paul R. Brakeman, Jiancheng Tu, Shouyang Yu, Ronald S. Petralia, Zhe Li, Ping–Wu Zhang, Joo Min Park, Xinzhong Dong, Bo Xiao, Paul F. Worley
Contents
Supplementary Figures 1–13
Nature Neuroscience: doi:10.1038/nn.3103
1 100
Human (1) --------------------------------------------------
Rat (1) --------------------------------------------------
Mouse (1) --------------------------------------------------
Chicken (1) --------------------------------------------------
Zebrafish (1) --------------------------------------------------
Drosophila (1) MLLPSGRSNSDVYPLQNVNTTTASASSVGGSKPLLLTRTSSPQLTTLQTL
--------------------------------------------------
--------------------------------------------------
----------------------------------------------------------------------------------------------------
--------------------------------------------------
NGGGIGGGGGANAHTALRTNSSAAFAATLAQETAGSLPGTGTQATATTST
101 200
Human (1) --------------------------------------------------Rat (1) --------------------------------------------------
Mouse (1) --------------------------------------------------
Chicken (1) --------------------------------------------------
Zebrafish (1) ---------------------------------------------MITVC
Drosophila (101) TASATTTTTAISTTTTAISTTATATATTAAASVNAVNTDFGAGALYGPIL
-------------------- S
---------------------------------------MRSHSCHRTKS
---------------------------------------MRSHSCHRTKS
---------MEEAEMNPSEKLATSFKVIWFTCVGKG MR RTDHRNKS
NWVINSSSSFFTRSSFPPQPFVPQCHCSPPPKLQIGRTFER RDKS
GPSHAGNAPIGNWGEIDRHLDAVQEKLKAGWTVHLAKDGRLYYCNHLTQS
201 300
Human (20) SG-----------------WPPPSGTWGLSQVPPYGWEM TANRDGRDYFI
Rat (12) SG-----------------WPPPSGTWGLNQVPPYGWEMMTNRDGRDYFI
Mouse (12) SG-----------------WPPPSGTWGLNQVPPYGWEMMTNRDGRDYFI
Chicken (42) SG-----------------WPHPSGTWSLNQGTPYGWEMTANRDGRDYFI
Zebrafish (56) FPRLYIVFTSTVLCLNGE CQTCVSNVAVMKEESKSCVICPHGKKKYRSIL
Drosophila (201) SG-----------------WLPACEDWSKHEELSYGWE RAIDSKGRSYYI
NHMTQAIPFDDPRLESCQIIPPAPRKVEMRRDPVLGFGFVAGSEKPVVVR
NHMTQAIPFDDPRFDSCQIIPPAPRKVEMRRDPVLGFGFVAGSEKPVVVR
NHMTQAIPFDDPRFDSCQIIPPAPRKVEMRRDPVLGFGFVAGSEKPVVVRNHMTQSATFEDPRIESCQITPPAPRKVEMRRDPVLGFGFVAGSEKPVVVR
HHLSQSSSLEEVRVDVEKFVPPAPRKVEMRRDPVLGFGFVAGSE KPVVVR
NHLNKTTTYEAPECIRWDEHPPEPRLVQLQRSASLGFGFVAGSERPVIVR
301 400
Human (103) SVTPGGPSEGKLIPGDQIVMINDEPVSAAPRERVIDLVRSCKESILLTVI
Rat (95) SVTPGGPSEGKLIPGDQIVMINDEPVSAAPRERVIDLVRSCKESILFTVI
Mouse (95) SVTPGGPSEGKLIPGDQIVMINDEAVSAAPRERVIDLVRSCKESILLTVI
Chicken (125) SVTPGGPSEGKLIPGDQIIMINDEPVSTAPRERVIDLVRSCKESILLTVV
Zebrafish (156) SVTPGGPSEGKLIPGDEIVMINEEPVSSAPRERVIDLVRSCKESIILTVV
Drosophila (284) FVTEGGPSIGKLQPGDQILAVNGEDVKDAPRDHVIQLVRACETQVSLLVC
QPYPSPKS-----AFISAAKKARLKSNPVKVRFSEEVIIN-----GQVSE
QPYPSPKS-----AFISAAKKARLKSNPVKVRFSEEVIIN-----GQVSE
QPYPSPKS-----AFISAAKKARLKSNPVKVRFSEEVIIN-----GQVSE
QPYPSPKS-----AFISAAKKARLKSNPVKVRFSEEVIIN-----GQVSE
QPYPSPKS-----AFISAAKKAKLKSNPVKVRFAEEVIIN-----GQVPE
QPMAHCVLPGRKSTLLSAGKRAKLRTRPSRVRFAESVCVNGAPLFPPSAF
401 500
Human (193) TVKDNSLLFMPNVLKVYLENGQTKSFRFDCSTSIKDVILTLQEKLSIKGI
Rat (185) TVKDNSLLFMPNVLKVYLENGQTKSFRFDCSTSIKDVILTLQEKLSIKGI
Mouse (185) TVKDNSLLFMPNVLKVYLENGQTKSFRFDCSTSIKDVILTLQEKLSIKGI
Chicken (215) TVKDNSLLFMPNVLKVYLENGQTKSFRFDCSTSIKDVILTLQEKLSIKCI
Zebrafish (246) SVKDNFLLFMPNVLKVFLENGQTKSFKFDCNTSIK---------------
Drosophila (384) SLGDICVPPMANVLKVFLENGQTKSFKYDATTTVQDVVSSLLDKLCLCCG
EHFSLMLEQRTEGAGTKLLLLHEQETLTQVTQRPSSHKMRCLFRISFVPK
EHFSLMLEQRTEGAGTKLLLLHEQETLTQVTQRPSSHKMRCLFRISFVPK
EHFSLMLEQRIEGAGTKLLLLHEQETLTQVTQRPSSHKMRCLFRISFVPK
EHFSLMLEQRVEGSGTKLLLLHEQETLTQVTQRPSSHKMRCLFRISFVPK
--------------------------------------------------
ELFSLVLEHVKSLKRNKLTLLDPQESLARIAARPGAHKLRCLFRITFVPI
501 600
Human (293) DPIDLLRRDPVAFEYLYVQSCNDVVQERFGPELKYDIALRLAALQMYIAT
Rat (285) DPIDLLRRDPVAFEYLYVQSCNDVVQERFGPELKYDIALRLAALQMYIAT
Mouse (285) DPIDLLRRDPVAFEYLYVQSCNDVVQERFGPELKYDIALRLAALQMYIAT
Chicken (315) DPIDLLRRDPVAFEYLYVQSCNDVVQERFGPELKYDIALRLAALQMYIAT
Zebrafish (281) --------------------------------------------------
Drosophila (484) SAAELAQRDLNALDYLYMQCCNDVNQERFAPELQPELALRLAALHMHQHA
VTT-KQTQKISLKYIEKEWGLETFLPSAVLQSMKEKNIKKALSHLVKANQ
VTT-KQTQKISLKYIEKEWGLETFLPSAVLQSMKEKNIKKALSHLVKANQ
VTT-KQTQKISLKYIEKEWGLETFLPSAVLQSMKEKNIKKALSHLVKANQ
VTT-KQTQKISLKYIEKEWGLETFLPSAVLQSMKEKNIKKALSHLVKANQ
-----------------EWGLAQFLPPAVLSSMKEKNIKKALTHILKTNQ
LANNFAPAKLTVKLVEREFGLERFVPVSLFEGMKRKELRRLISHFLKVNA
601 700
Human (392) NLVPPGKKLSALQAKVHYLKFLSDLRLYGGRVFKATLVQAEKRSEVTLLV
Rat (384) NLVPPGKKLSALQAKVHYLKFLSDLRLYGGRVFKATLVQAEKRSEVTLLV
Mouse (384) NLVPPGKKLSALQAKVHYLKFLSDLRLYGGRVFKATLVQAEKRSEVTLLVChicken (414) NLVPPGKKLSALQAKVHYLKFLSDLRLYGGRVFKATLVQGEKRSEVTLLV
Zebrafish (314) NLVPPGKKLTALQAKVHYLKYLSDLRLYGGRVFKSTLVQGEKHTEVTLLVDrosophila (584) EMTGSSKVLTQLQAKLHYLDIIASLPSYGAKCFSTNQREG---MERVLLV
GPRYGISHVINTKTNLVALLADFSHVNRIEMFSEEESLVRVELHVLDVKP
GPRYGISHVINTKTNLVALLADFSHVNRIEMFTEEESLVRVELHVLDVKP
GPRYGISHVINTKTNLVALLADFSHVNRIEMFTEEESLVRVELHVLDVKP
GPRYGISHVINTKTNLVALLADFSHVNRIEMFTEDESSVRVELHVLDVKP
GPRYGISHVINTKTNLVALLADFSHVNRIEMYTEDERNVRVELHVLDVKP
SPRFGLS QISSARNSVPQPISAIEDFTHVVVHREDDITCSVSIYMLGDRA
D-domain
Figure S1
R
R
KLSSHRTK
DGSR
IA-----------MDVFSFVK
AIA
Nature Neuroscience: doi:10.1038/nn.3103
701 800
Human (492) ITLLMESSDAMNLACLTAGYYRLLVDSRRSIFNMANKKNTATQETGPENK
Rat (484) ITLLMESSDAMNLACLTAGYYRLLVDSRRSIFNMANKKNAGTQDTG TENK
Mouse (484) ITLLMESSDAMNLACLTAGYYRLLVDSRRSIFNMANKKNAGTQDTG SENK
Chicken (514) ITLLMESSDAMNLACLTAGYYRLLVDSRRSIFNMANKKNAGNRETGIENR
Zebrafish (414) ITLIMESSDAMNLACLTAGYYRLLVDSRRSIFNMANN----SNTAGHDTR
Drosophila (681) VKFIMEDRDACEFSLVLGGYYRLLTG---NVLNVLRERE PNEEDNAPAFL
GKHNLLGPDWNCIPQMTTFIGEGEQEAQITYIDSKQKTVEITDSTMCPKE
GKHNLLGPDWNCMPQMTTFIGEGEQEAQITYIDSKQKAVEMTDSTLCPKE
GKHNLLGPDWNCMPQMTTFIGEGEQEAQITYIDSKQKTVEMTDSTLCPKE
GKHNLLASE WNCVPKTTTFLADGDQETQMPFGDPKQKAIDVSESLLCQKE
IKHNYQAID WNYG--T----CGGCNEPHPEYAVCGRRECDISPSEIHQAH
SQHTVLPAS WSYLLPFHNRAHSVNFLITPPYHPMPQQAVLSQPQPVQQQQ
801 900
Human (592) HRHLYIDNAYSSDGLNQQLSQPGEAPCEADYRSLAQRSLLTLSGPETLKK
Rat (584) HRHLYIDNTYSSDELSQPLTQPGDAPCEADYRSLAQRSLLTLSGPDTLKK
Mouse (584) HRHLYIDNSYSSDELNQPLTQPGDAPCEADYRSLAQRSLLTLSGPDTLKK
Chicken (614) HRHLYIENTYNSGGFDQHLTKQDPTDAEES-RNFNQHSLLSLSGLESSKK
Zebrafish (504) HMHGDEKAERQSRGSHMHAQPYFNIPKGKP--------------------
Drosophila (778) QLSYATDLDLHSVMATELLEEAAKDSGLSDSSGSNYCSESRTQLAVEA KN
AQESPRGAKVSFIFGDFALDDGISPPTLGYETLLDEGPEMLEKQRNLYIG
AQESPRGAKVSFIFGDLALDDGMSPPTLGYERMLDENPEMLEKQRNLYIS
AQESPRGAKVSFIFGDLALDDGMSPPTIGYERMLEENPEMLEKQRNLYIS
AQDSPRGAKVSFIFGDHNLDG-INPQTLGYERLLDESPEVLEKQRAIYIN
Q-ESPRSAKVSFIFGDPPLDS-VNPQNLGYHRLMEDIPEVLEEHR-YLYA
EQVLRRVQELQKLVQCSEQYLSEQEQELQHVPLQQLQQQQREQTQQQMGQ
901 1000
Human (692) SANDMKGLDLTPEAEGIQFVENSVYANIG--DVKSFQAAEGIEEPLLHDI
Rat (684) SANDMKNLDLTPDTDSIQFVANSVYANIG--DVKNFEAPEGIEEPLLHDI
Mouse (684) SANDMKNLDLTPDTDSIQFVANSVYANIG--DVKNFEAPEGIEEPLLHDI
Chicken (712) NANDIKGLELSPDAESIQFATNAVYATIN--DGKIFGAAEGIEEPLLHDI
Zebrafish (581) QQKDYKPLEASLEVDGFQYGTHMVYG-----DAKIFGTTEGIEEPLLHDI
Drosophila (878) SVAMLEFDSDCDSLNSSKVSSTEESNHVSQPSLKHSDSLTLLAETISHEL
CYAENTDDAEDEDEVSCEEDLVVGEMNQP----------AILNLSGSSDD
CYAENTDDAEDEDEVSCEEDLVVSEINQP----------AILDLSGSSDD
CYAENTDDAEDEDEVSCEEDLVVGEMNQP----------AILDLSGSSDD
CYAENTDDAEDEDEVSCEEDIMVGEINRP----------ALLSLSGSSDD
CYAETTDDAEEDDDISCEEDMMM MMGEMK------DKANSLLSLSESSDD
TGITQGLNAIPEPISASAPPTPA TPKRKPSLCSTS-SPRPQRKLNGFSQL
1001 1100
Human (792) IIDLTSLPPPE--GDDNEDDFLLRSLNMAIAAPPPGFRDSSDEEDSQSQA
Rat (772) IIDLTTLPPPE--GDDNEDDFLLRSLNMAIAAPPPGFRDSSDEEDTQSQA
Mouse (772) IIDLTTLPPPE--GDDNEDDFLLRSLNMAIAAPPPGFRDSSDEEDTQSQA
Chicken (800) IIDLTSLPPPE--GDDNEDDFLLHTLNMAIAAPPPGFRDSSDEEDSQNQA
Zebrafish (670) IIDLTSLPPPPEGDYEEDSDDLLESLNLAIAAPPPGFRDSSDEDDHHDRK
Drosophila (977) LSDLQALGTDFSQSESDSESVASPAQSPTTRRPQQLLQAGSKQQQQQQQH
ASFPEDK---EKGSSLQNDEIPVSLIDAVPTSAEGKCEKGLDNAVVSTLG
TSFHENK---EQGSSLQNEEIPVSLIDAVPTSAEGKCEKGLDPTVVSTLE
TSFHEDK---EQGSSLQNEEIPVSLIDAVPTSAEGKCEKGLDPAVVSTLE
TQPRDDK---EQASNLGSDDIPVSLIDAVPTNTEGKCEKGLDDAVVSTLQ
MSKN---------------DIPVSLIDSVPTQVAGGSEEVFN DAVVSTLQ
QQLAQRSSFGLHSPDGTHFGAETKDYNLREYLQQLKEISNVP DTDVAARQ
1101 1200
Human (875) ALEALSVSEEQQTS DNSG---VAILRAYSPESSSDSGNETNSSEMTESSE
Rat (867) ALEALSE--EQQKSENSG---VAILRAYSPESSSDSGNETNSSEMTEGSE
Mouse (867) ALEALSE--EQQKSENSG---VAILRAYSPESSSDSGNETNSSEMTEGSE
Chicken (895) ALEALAASEEQQTNDNSG---VAILRAYSPESSSDSGNETNSSEMTESSE
Zebrafish (755) ALEALAASEDQSHPQSDNSSGVEISRSFSPESSSDSGNETNSSEMTESSE
Drosophila (1077) LSEIYGFEVREDTFIETDTDLIDLRAIPPPQTPDELDSLSLLNAAPPKGF
LATAQKQSENLSRMFLATHEGYHPLAEEQTEFPASKTPAGGLPPKSSHAL
LAAAQKQSESLSRMFLATHEGYHPLAEEQTEFPTSKTPSVGLPPKSSHGL
LAAAQKQSESLSRMFLATHEGYHPLAEEQTEFPTSKAPSVGLPPKSSHGL
LAAAQKHSENTARMFLTTSEGYQPLVEEQTEFPIAKSQAGGPGMKSSQPL
LAAAQKLSENSLKMFVATAEGYQTLAEEETEFRLAPSLDDPQSS------
EGKAEELDSFLQQMLVAPPTQKATPAKELTPEEIMSFIIPPPPNLEAQPE
1201 1300
Human (972) AAR---PATDLPPKVVPSKQLLHSDHMEMEPETMETKSVTDYFSKLHMGS
Rat (962) AAR---PATDLPPKVVPSKQILHSDHMEMEPETMETKSVTDYFSKLHMGS
Mouse (962) AAR---PATDLPPKVVPSKQILHSDHMEMEPETMETKSVTDYFSKLHMGS
Chicken (992) AGC---QAADLQSKVVPSKQILHSDNMEMEPETMETKSVTDYFSKLHMGS
Zebrafish (849) -----------------AVANLRSDHFEMEPETMETKSLTEYFSKMHRDV
Drosophila (1177) RPEECLYSNSVQAKREFFQAVANGNNSIQQNNHKDQAAHVIEYPTVERKS
VAYSCTSKRKSKLADGEGKAPPNGNTTGKKQQGTKTAEMEEEASGKFGTV
VAYSCTSKRKSKLAEGEGKCPLSGNVPGKKQQGTKIAETEEDTKGKVGTV
VAYSCTSKRKSKLPEGEGKSPLSGNIPGKKQQGTKIAEIEEDTKGKAGTVLVYSCTSKRKSKMTESEVKAPSDGNATVKKQQGTKKAETDEELKAKFGTI
MRGMAQGKVEEQDS GMKSSAEAAARQLN--------IAESEDLVGMYSNF
KFSCCPTSKKEEAA SLEEAAPLQPPPRTPAIEQTTPPPARPPKSAELLQR
1301 1400
Human (1069) SSRDSQHLSTFNLERTAFRKDSQRWYVATEGGMAEKSGLEAATGKTFPRA
Rat (1059) SSRDNPHLSTFNLERTAFRKDSQRWYVASDGGVVEKSGVEAPAMKAFPRG
Mouse (1059) SSRDNPHLSTFNLERTAFRKDSQRWYVASDGGVVEKSGMEAPAMKVFPRG
Chicken (1089) SARDSQRLSTFNVERTAFR---QRWYGADDGAADKPS-PETANGKTFPRV
Zebrafish (924) NGRDSQCSAPFDMESMSYAFQ---------------------GSNQRLHM
Drosophila (1277) YSPKKQVRIVGHQPVVSVPLRELCPPQLPPRSSPTASSEPATPQTATPAT
SGLGAREAEGKEEGAPDGETSDGSGLGQGDRFLTDVTCASSAKDLDNPED
PGLGNREAEGKEDGTMEGEADDASGLGQGERFLSDMACVASAKDLDNPED
PGLGNREAEGKEDGTVEGGADDASVLGQGDRFLTDMACVASAKDLDNPED
PVRGKTETDCKDEVDPEVDQDDTSGLSQGENFLSDMTPVSSAKDLNDAED
ILNNKSEEHIYSEVAGDSAPLHADRVAQKVSLEDSIDLNASSPAKEQYLV
SAVSAPKKPPLPPIANRPRPVNGTPTPVSNTSPPIPATTRLPNAHSNGGQ
1401 1500
Human (1169) ADSSTCDHPSKLPEADESVARLCDYHLAKRMSSLQSEGHFSLQSSQGSSV
Rat (1159) TDSPTCDHATKLPEAEDNVARLCDYHLAKRMSSLQSEGHFSLQSSQGSSV
Mouse (1159) TDSPSCDHATKLSEAEDNVARLCDYHLAKRMSSLQSEGHFSLQSSQGSSV
Chicken (1185) TDLSADDHPSKLPEAEQSVARLCEYHLAKRMSSLQSEGHFSLQSSQCSSV
Zebrafish (1003) ERARSEHQQSKGASSEQEVTRLYEYHLSKRMSSLQSE GVHSLQSSQCSSI
Drosophila (1377) PTLPKKPQQLHGEKLFIKNGHLIDGEALLAKTDVAMA GLLIKLDQVAAQC
DAGCGTGSSGSACATPVESPLCPSLGKHLIPDASGKGVNYIPSEERAPGL
DTGCGPGSSSSACATPVESPLCPSMGKHLIPDASGKGGSYISPEERVAGH
DTGCGPGSSSSACATPVESPLCPSMGKHMIPDASGKGGRYISPEERAPGH
DAGCSTG--SSTCATPVESPLCTSDIKHVISDPSMKGIAYIPADERAAIL
DAGCSTG--SSSCVTPMDSPLCTVDSVHLHTESSMKGLGYTNQNVDGTFL
SAAQSAGGGTSIDEDKFQRARNELTEQTLALVTASKFLVASMSDMTLINL
Homer site
Nature Neuroscience: doi:10.1038/nn.3103
1501 1550Human (1269) PNHGATFKELHPQTE-----------------------------------
Rat (1259) PNHGATFKELHPQTE-----------------------------------
Mouse (1259) PNHGATFEELHPQTE-----------------------------------
Chicken (1283) SNHGTTYKDLHQQPE-----------------------------------
Zebrafish (1101) RKHHGQAGAESGREGSQRLPKIRETTVVFFIATVYMLSSPCLVDICSDAA
Drosophila (1477) PDHLTSCLTAIRRITELAQDMTR---------------------------
-----------------------------GMCPRMTVPALHTAINTEPLF
-----------------------------GMCPRMTVPALHTAINADPLF
-----------------------------GMCPRMTVPALHTAINADPLF
-----------------------------AVCHRMTVPVTHSAINAEPLF
DGDKRGFALTECPSSSSSRAQLVLCQARGDGTSRMLPCLLVHSASVHPLW
-------------------------HTSAP LQTRNIVLKVHDVASSFREL
1601 1637
Human (1305) GTLRDGCHRLPKIKETTV-------------------
Rat (1295) GTLRDGCHRLPKIKETTV-------------------
Mouse (1295) GTLRDGCHRLPKIKETTV-------------------
Chicken (1319) GTLREGCHRIPKIKETTV-------------------
Zebrafish (1201) LPQGGGKGILNKTTMGSGSIHTFYGNEMAVIIQAVVR
Drosophila (1525) VGVQIGPIGAGQLALQAECLANVLATLLRSLRVFSP-
Supplementary Figure 1: The amino acid sequence alignment of Preso1 from various creatures. Red residues shaded in yellow are identical in all the predicted proteins, dark blue residues shaded in light blue are identical in > 50% of the predicted proteins, similar residues are shown in black shaded in green. D–domain and Homer binding site are indicated.
Nature Neuroscience: doi:10.1038/nn.3103
101 200
hPreso1 (101) PGGPSEGKLIPGDQIVMINDEPVSAAPRERVIDLVRSCKESILLTVIQPY
hPreso2 (77) PGGPSENKLLAGDQIVAINEEDVSEAPRERLIELIRSAKEFIVLTVLHTH
PSP-KSAFISAAKKARLKSNPVKVRFSEEVIINGQVSETVKDNSL
FMPN
hPreso3 (81) AGGSAHGKLFPGDQILQMNNEPAEDLSWERAVDILREAEDSLSITVVRCTSGVPKSSFLTEEKRARLKTNPVKVHFAEEVLISGHS----QGNS
CMPN
QSP-KSAFISAAKKAKLRSNPVKVRFSEQVAVGETDAKMMKKEA
LIPN
201 300
hPreso1 (200) VLKVYLENGQTKSFRFDCSTSIKDVILTLQEKLSIKGIEHFSLMLEQRTE
hPreso2 (176) VLKVFLENGQIKSFTFDGRTTVKDVMLTLQDRLSLRFIEHFALVLEYAGP
GAGTKLLLLHEQETLTQVTQRPSSHKMRCLFRISFVPKDPIDLL
DPVA
hPreso3 (177) VLKLYLENGQTKAFKFEANTTVKDIILTVKEKLSIRSIEYFALALEEQYSIS--RLHLLHEEELIQQVVEREESHDYRCLFRVCFVPKDPLDLLK
EDPVA
EQNHKFLLLQDKQPLAYVVQRTHYHGMKCLFRISFFPKDPVELL
DPAA
301 400
hPreso1 (300) FEYLYVQSCNDVVQERFGPELKYDIALRLAALQMYIATVTTKQTQKISLK
hPreso2 (276) FEYLYIQSRNDVIRERFGMDPKPEMLLGLAALHIYITVSATRPSQKISLK
YIEKEWGLETFLPSAVLQSMKEKNIKKALSHLVKANQNLVPPGKK
--LSA
hPreso3 (275) FEYLYLQSCSDVLQERFAVEMKCSSALRLAALHIQERIYACAQPQKISLKYIEKDWGIENFISPTLLRNMKGKDIKKAISFHMKRNQNLLEPRQK
QLISA
NVEKEWGLEPFLPPSLLQVIKEKNLRKSLSQQLKAHQTHPSCGTK
--GSA
401 500
hPreso1 (400) LQAKVHYLKFLSDLRLYGGRVFKATLVQAEKRSEVTLLVGPRYGISHVIN
hPreso2 (376) IQAKLQYLRILNELPTFTGVLFNTVGLD- KQSATTLLVGPRHGISHVID
TKTNLVALLADFSHVNRIEMFSEEESLVRVELHVLDVKPITLLME
SSDAM
hPreso3 (375) AQLRLNYLQILGELKTYGGRIFNATLMLQDRESYIALLVGAKYGISQVINSKLNIMSTLAEFANISRVELTEESEKVSVVKVYLQDVKVLTLLLE
SNSAK
LKTNLTTVLSEFSKISKIQLFRENQGVARVETSIMDAKPLVLLME
WPEAT
501 600
hPreso1 (498) NLACLTAGYYRLLVDSRRSIFNMANKKNTATQETGPENKGKHNLLGPDWN
hPreso2 (473) NFACLIAGYCRLLLDSRKMVFSRPASQPLPPPMIKADYMHSAHRPVTGGH
CIPQMTTFIGEGEQEAQITYIDSKQKTVEITDSTMCPKEHRHLYI
DNAYS
hPreso3 (475) DLACLIAGYYRLLVDPVTSIFLWPGNKQQAHRVSAEEGYESRACSDSEESSEVDCVLEPLSDRRLVKLAPCRSLIK-------------------
EEQPP
LGKKESSYVG-----------------------------------
S
601 700
hPreso1 (598) SDGLNQQLSQPGEAPCEADYRSLAQRSLLTLSGPETLKKAQESPRGAKVS
hPreso2 (538) VGTSPRKSSRCTPPPADSELVSFCYLHMREQ------RKEQESRTDVNEN
1 100
hPreso1 (1) FVKIAK HRTKSSGWPPPSGTWGLSQVPPYGWEMTANRDGRDYFINHM
hPreso2 (1) ---MET QRVQDRLLAAPGCSSPSGQQELFSSHVMQEES---------
TQAIPFDDPRLESCQIIPPAPRKVEMRRDPVLGFGFVAGSEKPVV
VRSVT
hPreso3 (1) MEELETSLFQTRKAHRIEQMVARWLRRSRDSSARAKVAAADG---------------PARNPTQTLIPVRHTVKIDKDTLLQDYGFHISESLPLT
VVAVT
-------ANDMECEQLPAEILRQVTVHRDPIYGFGFVAGSERPVV
VRSVR
FIFGDFALDDGISPPTLGYETLLDEGPEMLEKQRNLYIGSANDMK
LT
hPreso3 (556) GNSPTPEVARRGPSTCGASSTTDSAESEASDSANTESRGYRTSGSSESMDALEEDDLDTCSSSRSTFFHFGSPGLAESIDSDSQEERSGIETSGF
LD
LIFFEETRPRTKSDPTS----------------------------
701 800
hPreso1 (698) PEAEGIQFVENSVYANIGDVKSFQAAEGIEEPLLHDICYAENTDDAEDED
hPreso2 (600) -KSSGQGYEVVPDDFDAASLDHEPCASRARSYTLDNSLGAEALNFYCDSC
EVSCEEDLVVGEMNQPAILN------------LSGSSDDIIDLTS
LPPP-
hPreso3 (656) LAQRANPQCQKTEFSESAALETFGWAPELSTVRLDPRLYEGSHADYYSLCSSVSPASYLSDSSESTASRQGGAPPAWGQQGWTEAQPSSMLEPLA
LHPPL
KAKLQEQLGPRKGGKPGSS-----------------RDNIVDLMS
LPPPG
801 900 hPreso1 (786) EGDDNEDDFLLRSLNMAIAAP--PPGFRDSSDEEDS-QSQAASFPEDKEK
hPreso2 (677) SEEEEEEEDETTSLLPAIAAP--PPGFRDNSSDEDDPKRRAVQSQEQGRH
G------SSLQNDEIPVSLIDAVPTSAEGKCEKGLDNAVVSTLGA
ALS
hPreso3 (756) AFEDGSSDEEYYDAADKLTPPGPPSGPRDVSTAEPSATSLQNKASTSSPENSLPCGPDGRQPSRRGGVKKYAKTLRKRRSFLQTDDTSQVSFPLV
PSASL
L------RGLLYDEIPVTLIDSVQTRTVRDHAQELDDALVSTLQA
ALA
901 1000
hPreso1 (876) VSEE---QQTSDNSGVAILRAYSPESSSDSGNETNSSEMTESSELATAQK
hPreso2 (768) ASEDGPHPPPPQTAGLIVLATITPESSLDSGHETNSSELTDMSEMMSAMK
QSENLSRMFLATHEGYHPLAEEQTEFPASKTPAGGLPPKSSHALA
ARPAT
hPreso3 (856) ESVDDVCYYDREPYLALGAPSPTVSSLQDMQGEPGLLETKALGLLAPLRETKSTNPASRVMEMEPETMETKSVIDSRVSSISAIRFRIDPNNKEN
SGVVP
QHQNTTYFLAQHLNKDSLLARKDLPFRIQSCAAQAVLTAPYSLGR
PDPNP
1001 1100
hPreso1 (973) DLPPKVVPSKQLLHSDHMEMEPETMETKSVTDYFSKLHMGSVAYSCTSKR
hPreso2 (868) SLQPIATGQSPGPPGARRKLPQSEGQVQGERTYSLAVHPALSPQLSEQKN
KSKLADGEGKAPP-----------NGNTTGKKQQGTKTAEMEEEA
KFG
hPreso3 (956) AASSSASTPHCSNPGSSGPDTAQARPSQ-ILPLSQDLDGIAPKEPTIEHGDSSFSLSSGDPNPDRACLAS----NPGLNNVSQGDTLELQLEPHV
LEMG
LSLLSPVPEDKGPGHTRAGLEMSLRAATSSLSEEQVSELRDNLPK
VRLS
1101 1200
hPreso1 (1062) TVSSRDSQHLSTFNLERTAFRKDSQRWYVATEGGMAE----KS--GLEAA
hPreso2 (968) PKLILDPKSSVTPAIISAALQQVVHNKSLVTAGGALGNPPSRGERRLEAS
TG------KTFPRASGLGAREAEGKEEGAPDGETSDGSGLGQGDR
hPreso3 (1052) LESFCTNHIQETAPKYTEPLLSPRDEPRSDECGINPGEK-IASIPTKEEPQGQLSLERDREVTNKNGTNVFQEESRKDSGDSPGDVSNNVSQTLD
MGRPEVSMMSSSASKNLKFKISPSAPETSWNSQHQLGAEVSSSPR
D-domain
E
Homer site
MDVFS LSS
LDS-----
-----
L
L
L
L
L R
R
R
R
----
GLD
LCL
-----
LE
LE
SG
Q
E
Figure S2
Nature Neuroscience: doi:10.1038/nn.3103
1201 1300
hPreso1 (1150) TCASSAKDLDNPEDADSSTCDHPSKLPEA--DESVARLCDYHLAKRMSSL
hPreso2 (1068) RADSLHLSQQEDSLPVQNFPPKSYLLRTS--RESVGKQATGEVAGKGGPV
QSEGHFSLQ--SS-QGSSVDAGCGTGSS------------G----
hPreso3 (1152) GKIVTSLSLDAPVTGTEQIPPHPPRDPQGQSREPPGQGCQAQEQKLFVELDLDPDFFLGKQTVSPAVPPEGIKAEAPNHVTGQDIAPRDSPEWVC
GGKPTLQKQGTISSQGEKAQLESTPKRSKLEETSLVPRATYPMAL
1301 1400
hPreso1 (1229) ---SACATPVESPLCPSLGKHLIPDASGKGVNYIPSEE-----RAPGLPN
hPreso2 (1166) ---PSCQSRSHSPSCQPHGHSPSSQSRGQSPSCQPRGQSPLRSQAASRQV
HGATFKELHPQTEGMCPRMTVPALHTAINTEPLFGTLRDGCHRLP
KIKET
hPreso3 (1252) SLPEPLPCPQEDPHLETSNHCLLSEGKSDSSSICLSAEKSFLCFAPESHPEVSASLRVATSLGFAGMNEMVAPRIGMDQCSCQFSYATCFRGPQP
E----
STMPSRKLETTLNGAHSTSEGPAKPKSSRGPFRLRNLFSATFPTR
KET
1401 1500
hPreso1 (1316) TV------------------------------------------------
hPreso2 (1260) DERQAQLQKVKQYELEFLEELLKPPSQGELPGTEYLQPPAPGRCSCQLRS
---------------------------------------------
hPreso3 (1352) TEEEDR--DLEAHPMAPLTS---PPSAGSPVVLPWRPARAHSCTTAPLSR KSHIWPEYCSRALRQLKATPASTPEGFIQLMESLLELQDILETSW
VGNK
SPVQQGPGMSREQRRSCDCKRICRGGRPQATQTPVPSLRGRERDR
LPSQ
1501 1600
hPreso1 (1316) --------------------------------------------------
hPreso2 (1360) RQPEAGPGVSLSSPINVQRIRSTSLESRECRSDPESGVSCLTTCASGGEC
---------------------------------------------
hPreso3 (1443) HPPEKCTWHFTESRSRLCMGSQKLLSSCRHVIRMDQSPEEMQGAVRDTFQ HLVQLAGLCFQFTDCSRCSARHREAAGNLRDVVYTYHQFIEAAKS
LGAPNYRKLMRRYSISELDQGDRASLTSDVYPHPPLGMLPREAKE
1601 1700
hPreso1 (1316) --------------------------------------------------
hPreso2 (1460) PIALGPKSRSLESPTLGDPSYVQVAPETKGPRQMAVFSLPEEVYRKPAEL
---------------------------------------------
hPreso3 (1541) YHDLSVKLLARQCTALTAAVFCLTQKFRASTAL----------------- ---------------------------------------------
DEDSESSKCCSIRYCFYYRKCDMADDASDGKDELSYSIPMKILPG
1701 1800
hPreso1 (1316) --------------------------------------------------
hPreso2 (1560) QVVPVVSRTLQVLDAATCSSSSPEASRTQEIDLRVSTFEGSLAKINALRA
---------------------------------------------
hPreso3 (1541) -----------------------------------------------------------------------------------------------HAYGLPDGFLAARLDTNELLTVLRQCVASPEARAPKPYVSQISEY
1801 1900hPreso1 (1316) --------------------------------------------------
hPreso2 (1660) LKFKELRASCRRVANVDKSPTHMLAAITGSFQVLSSLIETFVRLVFIVRS
---------------------------------------------
hPreso3 (1541) -----------------------------------------------------------------------------------------------
EAQRQELLAKVEEVVRNYTFLLRAAEESTARNLNQQQQQQQQQQQ
1901 1950
hPreso1 (1316) ---------------------------------------------
hPreso3 (1541) ---------------------------------------------hPreso2 (1760) QQQQQQQQQQQVAAAAGAATEHPPGSPTSATVMSTFTHSLKTLIK
FLTDV
ISSPA
APTGS
-----
FNPEP
QS---
QK
G
V
-----
TCERG
VEASL
-----
-----
-----
-----
-----
-----
-----
MKLDE
KLELA
QQQQQ
Supplementary Figure 2: The amino acid sequence alignment of human Preso family. Red residues shaded in yellow are identical in all the predicted proteins, dark blue residues shaded in light blue are identical in > 50% of the predicted proteins, similar residues are shown in black shaded in green. D–domain and Homer binding site are indicated.
Nature Neuroscience: doi:10.1038/nn.3103
Preso1
Preso3
Preso2
Supplementary Figure 3: Preso family expression patterns by in situ hybridization. 1Kb C–terminal coding region of each gene was used for antisense and sense probes that were labeled with [35S].
Figure S3
Nature Neuroscience: doi:10.1038/nn.3103
aMyc–Preso1: + +Myc–Preso2: + +Myc–Preso3: + +Myc–Homer1c: + + +
Lysate
IP: Homer1c
Blot with anti–myc
mGluR2: + + + +Myc–Preso1: + +
mGluR4: + + + + Myc–Preso1: + +
HA–mGluR5: + + + +Myc–Preso1: + +
anti–HA
anti–Myc
anti–mGluR2
anti–Myc
anti–mGluR4
anti–Myc
b
Lysate IP: anti–Preso1
Lysate IP: anti–Preso1 Lysate IP: anti–Preso1c d
98KD
49KD
98KD
49KD
188KD
98KD
188KD
98KD
188KD
98KD
pS–mGluR
mGluR5 50 KD
50 KD
mGluR5 C–term:
λ–Phosphatase: – – + +S11
26A
WT S11
26A
WT
e
Figure S4
Nature Neuroscience: doi:10.1038/nn.3103
Supplementary Figure 4: Preso family: co–IP assays with Homer and mGluRs. a, Preso1 and Preso2 co–IP with Homer1c, but Preso3 does not. Myc–Homer1 and Myc–Presos were transfected into HEK293T cells. Detergent lysates of the cells were incubated with anti–Homer1c antibody and then analyzed by Western blotting with anti–Myc antibody. b, mGluR5 co–IPs with Preso1. Myc–Preso1 and HA–mGluR5 were transfected into HEK293T cells. Detergent lysates were incubated with anti–Preso1 antibody and blotted with anti–Myc and anti–HA antibody. c,mGluR2 does not co–IP with Preso1. Myc–Preso1 and mGluR2 were transfectedinto HEK293T cells. Detergent lysates were incubated with anti–Preso1 antibody and precipitates analyzed by Western blotting with anti–Myc and anti–mGluR2 antibody. d, mGluR4 does not co–IP with Preso1. Myc–Preso1 and mGluR4 were transfected into HEK293T cells. Detergent lysates were incubated with anti–Preso1 antibody and precipitates analyzed by Western blotting with anti–Myc and anti–mGluR4 antibody. e, Characterization of mGluR5 phosphorylation antibody. The phosphorylation site–specific anti–mGluR5–pS1126 antibody detects wild–type mGluR5 C–terminus from transfected HEK293 cells, but not mGluR5(S1126A) mutant. λ–phosphatase treatment of the immunoblot abolishes reactivity of wild–type mGluR5 C–terminus. A phosphorylation–independent anti–mGluR5 C–terminus antibody is used to detect expression of wild–type and mutant mGluR5 C–terminus.
Nature Neuroscience: doi:10.1038/nn.3103
EX2 EX3 EX490kb
Nhe I NheIShort Arm Long Arm
PGK-neo EX3
EX3
LoxP LoxPfrtfrt
Figure S5
353 bp517 bp
245 bp409 bp
+/+ +/– –/– +/+ +/– –/– +/+ +/– –/– +/+ +/– –/–
Cortex Cerebellum Cortex Cerebellumb
a
Preso1locus
Preso1 protein
Targeting vector
EX2 EX3 EX4
90kbPGK-neo
LoxP LoxPfrtfrt
EX2 EX4
90kbLoxPCre-recombinase expression
Preso1 floxedallele
Preso1- allele
c
Preso1
Actin
Preso1
Actin
WT
d
188KD
98KD
Cortex Hippocampus Striatum CerebellumW1 W2 K1 K2 W1 W2 K1 K2 W1 W2 K1 K2 W1 W2 K1 K2
Homer1
Homer2
Homer3
GluN1
GluA1
GluA2/3
Actin
e
WW PDZ FERMRA Homer Ligand PDZ Ligand
ETTVPPPGFRD
188KD
98KD
38KD
38KD
49KD
49KD
49KD
98KD
98KD
98KD
38KD
Preso1–/–
WT Preso1–/–
Nature Neuroscience: doi:10.1038/nn.3103
Supplementary Figure 5: PKAP1 knockout mice generation. a, Schematic representation of the Preso1 gene targeting strategy. The relevant portion of the Preso1locus Exon 3 was inserted in the LoxP/PGK–neo cassette to make a conditional knockout. CMV–CRE mice were employed to generate Preso1–/– mice. b, RT–PCR analysis after CMV–CRE expression. Two independent primer sets in different exons were used. c,Western blot analysis shows absence of Preso1 immunoreactivity in Preso1–/– mouse brain. d, Western blot analysis shows absence of Preso1 immunoreactivity in Preso1–/– cortical neurons. e, Western blot analysis showing no significant difference of Homer1, Homer2, Homer3, NMDA receptor GluN1, GluA1 and GluA2/3 protein expression between 8–week–old WT and Preso1–/– mouse brain. W: WT; K: Preso1–/–.
Nature Neuroscience: doi:10.1038/nn.3103
a bWT
WT/ MPEP
0-10min 10-60min0
50
100
150
200
250300
350
Dur
atio
n of
resp
onse
(s)
***
Figure S6
-10 0 10 20 30 40 50 60-20
0
20
40
60
80
100
Dur
atio
n of
resp
onse
(s)
Time after formalin injection (min)
400Grm5–/– / MPEP
Grm5–/–WT
WT/ MPEPGrm5–/– / MPEP
Grm5–/–
Supplementary Figure 6: Grm5–/– mice show reduced response to formalin–induced inflammatory pain. a, b, Formalin–induce inflammatory pain in Grm5–/– mice. a, The duration of behavioral responses to hind paw formalin injection in 5–min intervals in Grm5–/–mice and their WT littermates with or without MPEP (30mg/kg, i.p.) pre–injection. b, Results from a grouped into two phases. *P < 0.05, **P < 0.01, n = 7–11 for each group.
Nature Neuroscience: doi:10.1038/nn.3103
Figure S7
WT
0 200 400 600 800 1000
0
2
4
6
8
10
Spik
e nu
mbe
r
Injected current (nA)
WTa
b
20 ms
20 m
V
300 nA
Preso1–/–
Preso1–/–
Supplementary Figure 7: Excitability of DRG neurons is not different between WT and Preso1–/– mice. The small–diameter DRG neurons were cultured and injected with depolarizing currents stepwise from 100 to 900 nA with 100 nA interval. Each injected current lasted 200 ms and was separated by an interval of 4 s. The excitability was evaluated by the number of action potentials. WT and Preso1–/– DRG neurons do not show a significant difference of excitability. Eight neurons from 3 WT mice and 10 neurons from 3 Preso1–/– littermates were recorded.
Nature Neuroscience: doi:10.1038/nn.3103
Figure S8
ba
dc
fe
**
**
*WT
0-10min 10-60min0
100
200
300
400
500
600
**
Dur
atio
n of
resp
onse
(s)
WT
700
800
-10 0 10 20 30 40 50 60-20
020406080
100120140160180
Dur
atio
n of
resp
onse
(s)
Time after formalin injection (min)
*
WT
0-10min 10-60min0
100
200
300
400
500
600
**D
urat
ion
of re
spon
se (s
)
WT
-10 0 10 20 30 40 50 60-20
0
20
40
60
80
100
120
140
Dur
atio
n of
resp
onse
(s)
Time after formalin injection (min)
700
-10 0 10 20 30 40 50 60-20
0
20
40
60
80
100
120
Dur
atio
n of
resp
onse
(s)
Time after formalin injection (min)0-10min 10-60min
0
50
100
150
200
250
300
350
400 *
Dur
atio
n of
resp
onse
(s)
WT WT
Homer2–/–Homer3–/– Homer2–/–Homer3–/–
Grm5R/RGrm5R/R
Homer1a–/–Homer1a–/–
Nature Neuroscience: doi:10.1038/nn.3103
Supplementary Figure 8: Formalin–induced inflammatory pain responses inHomer2–/–Homer3–/– mice, Grm5R/R mice and Homer1a–/–mice. a, b, Formalin–induce inflammatory pain is increased in Homer2–/–Homer3–/– mice. a, The duration of behavioral responses to hind paw formalin injection in 5–min intervals in Homer2–/–Homer3–/– mice and their WT controls. b, Results from a, grouped into two phases. *P < 0.05, **P < 0.01, n = 12 and 8 for WT and Homer2–/–Homer3–/– mice. c, d,Formalin–induced inflammatory pain is increased in Grm5R/R mice. c, The duration of behavioral responses to hind paw formalin injection in 5–min intervals in Grm5R/Rmice and their WT littermates. d, Results from c grouped into two phases. *P < 0.05, **P < 0.01, n = 8 for each group. e, f, Formalin–induce inflammatory pain is reduced in Homer1a–/– mice. e, The duration of behavioral responses to hind paw formalin injection in 5–min intervals in Homer1a–/– mice and their WT littermates. f, Results from e, grouped into two phases. *P < 0.05, n = 9 for each group.
Nature Neuroscience: doi:10.1038/nn.3103
Figure S9
b cHA–Preso1: + F806R + +HA–Homer1c: + + G89N W24A
Lysate
IP: anti-Preso1
49KD
98KD
62KD
188KD
Homer1c
Preso1
Homer1c
Preso1
28KD
38KD
49KD
98KD
62KD
188KD
28KD
38KD 49KD
98KD
62KD
188KD
28KD
38KD
49KD
98KD
62KD
188KD
38KD
49KD
98KD
62KD
188KD
28KD
38KD
49KD
98KD
62KD
188KD
98KD
188KD
Homer3
IP: anti–Preso1
mGluR1
Lysate PI IP
IP3R
Shank
PSD95
Preso1
Supplementary Figure 9: Full length western blot for Figure 1. Samples were loaded on a 4–12% gel and blotted for the antibodies indicated in the figure. Dotted lines indicated that the bands were used for inclusion in Figure 1.
Nature Neuroscience: doi:10.1038/nn.3103
Figure S10a b
c e
98KD
62KD
188KD
Preso1 / Lysate
IP: anti–Preso1
HA–Preso1:
Myc–mGluR5: + + + + + + F1128R FL
ΔPDZ
ΔW
W
ΔFE
RM
mGluR5 / co–IP
mGluR5 / Lysate
Preso1 / IP
F806
R
F806
R
98KD
188KD
98KD
188KD
62KD
98KD
188KD
Myc–Preso1FERM: +
HA–mGluR5: + +
Preso1FERM / Lysate
IP: anti-Myc
mGluR5 / co–IP
mGluR5 / Lysate
Preso1FERM / IP
49KD
98KD
62KD
188KD
28KD
38KD
49KD
98KD
62KD
188KD
28KD
38KD
YVFYV FR W
THA–mGluR5:
IP: anti–Myc
98KD
188KD
98KD
188KD
188KD
188KDPreso1 / Lysate
mGluR5 / co–IP
mGluR5 / Lysate
Preso1 / IP
Myc–Preso1: + + + +HA–mGluR5:
Myc–Preso1: + + + +W
T1~
1020
1~92
0
IP: anti–Myc
98KD
188KD
188KD
98KD
Preso1 / Lysate
mGluR5 / co–IP mGluR5 / Lysate
Preso1 / IP
62KD
WT
1~10
20
1~92
0
Nature Neuroscience: doi:10.1038/nn.3103
Supplementary Figure 10: Full length western blot for Figure 2. Samples were loaded on a 4–12% gel and blotted for the antibodies indicated in the figure. Dotted lines indicated that the bands were used for inclusion in Figure 2.
Nature Neuroscience: doi:10.1038/nn.3103
Figure S11
a b
mGluR5
Homer1c:
Preso1
Homer1c
Lysate
+ ++ +
+
Homer1cIP
+ ++ +
+
Lysate
+ ++ +
+
pS–mGluR
188KD
98KD
mGluR5WT:
Preso1:
49KD
98KD
188KD
28KD
38KD
mGluR5
Homer1c:
Preso1
Homer1c
Lysate
+ ++ +
+
Homer1cIP+ ++ +
+
Lysate
+ +
+ +
Homer1cIP+ +
+ +++
mGluR5WT:mGluR5TSAA:
Preso1FR:
188KD
98KD
49KD
98KD
188KD
28KD
38KD
62KD
62KD
Supplementary Figure 11: Full length western blot for Figure 3. Samples were loaded on a 4–12% gel and blotted for the antibodies indicated in the figure. mGluR5 dimers were used for mGluR5 band quantification. Dotted lines indicated that the bands were used for inclusion in Figure 3.
Nature Neuroscience: doi:10.1038/nn.3103
Figure S12
a b c
eg
d
f
HA–mGluR5:
HA–CDK5/P35:
HA–MEK DD:
+ + ++
+
98KD
188KD
38KD
98KD
188KD
28KD
49KD
pS–mGluR
CDK5
mGluR5
P35
MEK DD
pS–mGluR
mGluR5
Con
trol
UO
126
Purv
alan
olA
98KD
188KD
98KD
188KD
+ + + +
Preso1
CDK5
+ +HA–CDK5:HA–Preso1:
IP: anti–Preso1 Lysate
98KD
28KD
38KD
188KD
Preso1
IP: anti–Preso1 Lysate
ERK1
+ + + +HA–ERK1:+ +HA–Preso1:
98KD
38KD
49KD
188KD
HA–mGluR5:
HA–Preso1:
HA–ERK1:
IP: anti–mGluR5 Lysate
Preso1mGluR5 monomer
mGluR5 dimer
ERK1
+ ++ +
+ + +
+ ++ +
+ + +
98KD
188KD
38KD
49KD
IP: anti–CDK5
Lysate PI IP
CDK5
Preso1
IP: anti–ERK
ERK1/2
Preso1
Lysate PI IP
49KD
98KD
62KD
188KD
38KD
28KD
38KD
49KD
98KD
62KD
188KD
28KD
38KD
Supplementary Figure 12: Full length western blot for Figure 4. Samples were loaded on a 4–12% gel and blotted for the antibodies indicated in the figure. mGluR5 dimers were used for mGluR5 band quantification. Dotted lines indicated that the bands were used for inclusion in Figure 4.
Nature Neuroscience: doi:10.1038/nn.3103
Figure S13
mGluR5 / Lysate
mGluR5 / co–IP
Homer / IP
Preso1 / Lysate
pS–mGluR
Cx Sc
WT Preso1–/– WT Preso1–/–
49KD
98KD
188KD
38KD
98KD
188KD
98KD
188KD
98KD
62KD
188KD
mGluR5 / IP
ERK1/2 / co–IP
ERK1/2 / Lysate
mGluR5 / Lysate
WT Preso1–/–
49KD
38KD
49KD
38KD
98KD
188KD
98KD
188KD
a
b
c
d
Ctl BD Ctl BD
mGluR5 / Lysate
mGluR5 / co–IP
Homer / IP
Ctl DH Ctl DH
Actin / Lysate
pS–mGluR
WT Preso1–/– WT Preso1–/–
49KD
98KD
188KD
38KD
98KD
188KD
98KD
188KD
49KD
38KD
mGluR5 / Total
mGluR5 / Surface
Actin / Surface
Actin / Total
Ctl D5 Ctl D5 Ctl D30 Ctl D30
WT Preso1–/– WT Preso1–/–
49KD
38KD
98KD
188KD
98KD
188KD
49KD
38KD
Nature Neuroscience: doi:10.1038/nn.3103
Supplementary Figure 13: Full length western blot for Figure 5. Samples were loaded on a 4–12% gel and blotted for the antibodies indicated in the figure. mGluR5 dimers were used for mGluR5 band quantification. Dotted lines indicated that the bands were used for inclusion in Figure 5.
Nature Neuroscience: doi:10.1038/nn.3103