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SUPPLEMENTARY INFORMATION

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doi: 10.1038/nature08966

Dm Calypso/ dBap1Hs BAP1Mm Bap1

UCH

UCH

UCH

111 728

729471

Supplementary Figure 1

b 4 GWLELESDPGLFTLLVEDFGVKGVQVEEIYDLQSKCQGPVYGFIFLFKWIEERRSRRKVSTLVDDTSVIDD Hs BAP1 4 GWLELESDPGLFTLLVEDFGVKGVQVEEIYDLQSKCQGPVYGFIFLFKWIEERRSRRKVSTLVDDTSVIDD Mm Bap1 45 GWLELESDPGLFTLLLKDFGCHDVQVEEVYDLQKPIESP-YGFIFLFRWIEERRARRKIVETTAEIFVKDE Dm Calypso ***************::*** :.*****:****. :.* *******:******:***: . : * *: 75 DIVNNMFFAHQLIPNSCATHALLSVLLNC--SSVDLGPTLSRMKDFTKGFSPESKGYAIGNAPELAKAHNS Hs BAP1 75 DIVNNMFFAHQLIPNSCATHALLSVLLNC--SNVDLGPTLSRMKDFTKGFSPESKGYAIGNAPELAKAHNS Mm Bap1115 EAISSIFFAQQVVPNSCATHALLSVLLNCNENNLQLGDTLSRLKTHTKGMSPENKGLAIGNTPELACAHNS Dm Calypso : :..:***:*::**************** ..::** ****:* .***:***.** ****:**** **** 144 HARPEPRHLPEKQN-GLSAVR-TMEAFHFVSYVPITGRLFELDGLKVYPIDHGPWGEDEEWTDKARRVIME Hs BAP1144 HARPEPRHLPEKQN-GLSAVR-TMEAFHFVSYVPITGRLFELDGLKVYPIDHGPWGEDEEWTDKARRVIME Mm Bap1186 HAMPQARRRLERTGAGVSSCRFTGEAFHFVSFVPINGQLFELDGLKPYPMNHGGWEDSEDWTDKFRRVMAE Dm Calypso ** *:.*: *: . *:*: * * *******:***.*:******** **::** * :.*:**** ***: *

213 RIGLATAGEPYHDIRFNLMAVVPDRRIKYEARLHVLKVNRQTVLEALQQLIRVTQPELI - 354 AA – Hs BAP1213 RIGLATAGEPYHDIRFNLMAVVPDRRIKYETRLHVLKVNRQTVLEALQQLIRVTQPELI - 353 AA – Mm Bap1257 RLGIATGEQ---DIRFNLMAVVPDRRIAITHKLKMLRTNQAIVSGTLQKLLKA------ - 27 AA - Dm Calypso *:*:**. : *************** :*::*:.*: * :**:*::.

626 KYSPKELLALLKCVEAEIANYEACLKEEVEKRKKFKIDDQRRTHNYDEFICTFISMLAQEGMLANLVEQNI Hs BAP1625 KYSPKELLALLKCVEAEIANYEACLKEEVEKRKKFKIDDQRRTHNYDEFICTFISMLAQEGMLANLVEQNI Mm Bap1334 ---ARDLQSLLKNLDTEIAINEQHLADENDRRHMFKVDASRRTHNYDKFICTFLSMLAHQGVLGELVSQHL Dm Calypso .::* :*** :::*** * * :* ::*: **:* .*******:*****:****::*:*.:**.*::

697 SVRRR---QGVSIGRLHKQ-----------------------------------RKPDRRKRSRPYKAKRQ Hs BAP1696 SVRRR---QGVSIGRLHKQ-----------------------------------RKPDRRKRSRPYKAKRQ Mm Bap1402 LPSKKVSGQGAA-NRISKQSTTASAGGSTAAGTASTPKTQQQQAAAAKNGKSPSKTPGRRRKGRNKCRKRK Dm Calypso :: **.: .*: ** :.*.**::.* **:

* and X = conserved residue : and X = conservative substitution . and X = semi-conservative substitution

and X and * = conserved catalytic residue

| start UCH domain

| end UCH domain

endogenousCalypso

TAP-Calypso*62

83wt

TAP-calypso

Δ

a

c

2009-07-08008Scheuermann et al.

Supplementary Figure 1. calypso (CG8445) encodes the ubiquitin C-terminal hydrolase (UCH) family member dBap1.

a. Schematic represenation of the Calypso/ dBap1 protein and its putative orthologs in Homo sapiens (Hs) and Mus musculus (Mm).

b. MAFFT alignment (v6.707b) in CLUSTAL format of Calypso (UniProt Q7K5N4) with human (Q92560) and mouse (Q99PU7) BAP-1. Three residues strictly required for activ-ity (C131, H213, and D228 in Dm Calypso) are conserved in all three proteins (arrows and marked in yellow). The UCH domain encompasses amino acids 45 to 261 in Dm Calypso/ dBap1 (SMART prediction).

c. Western blot of nuclear extracts from wild-type (wt) and α-tubulin1-TAP-calypso (TAP-calypso) transgenic embryos, probed with anti-Calypso antibody. Expression levels of the TAP-Calypso and endogenous Calypso proteins cannot be directly compared because the antibodies bind not only the Calypso epitope but also the Protein A moiety in the TAP-tag. Asterisk in wt lane indicates a cross-reacting protein; Δ indicates TAP-Calypso degradation products.


doi: 10.1038/nature08966 SUPPLEMENTARY INFORMATION

1 mktitpdttt ttssqhqqll ipqadqhhqp mlqqqsllaa ppptmimehv nlvdddekdp laleqlevsp stkhthslrr hlpriivkpi ppekkpmaps eeaavstapa pptrlicsrr

121 iqqqqqvkaa aaaaaaaaaa aaaaaaaaqa qatssypsai spgskagtsq astmrevlas ipgfsvkprr rsnkklttaa qieqtkdgki dletpdsila stnlrallnk qtfsllpply

241 qynliqllps vdreaseleq psssasggsp seairlsasc lnneffarac lewrerlseg eftpenqlkl kteaereknk ldpwklkhfe pfwgeknsrg kdkdklesdc knqklsasik

361 sepkppatsq qkplqqatcd netelkfdls tkcettsakt tvavavadks stfpptgsqn nvlneqqrrv lkrpssspsq rkqapttiat inldddldel pstskdskqp kmdeivpnas

481 gnvvaapmvd vvdhsavemk ikdeqqhqrq hqplinstcd kiepsecske mivamkqvds kedvdsiasa aampaiaavt phtpkpeala pnpdvanqfv sylqnvelaa etkapldnsn

601 eadittgtns hdfvfsdtid hayfqehqst inhnfftsss ssntattaan kleehsdkpe dsplpiassi sgstpassit stsctsssss sasmssscss snsgstttap ttsssagapt

721 apltlaaaae ttlanvqaml stvaklqqqq qelpvelnsn emyqhvqhdw nfgdiklsss qssgdqqrnl sheaidlmdv vqdadviddi mhndvchdvl gdedegdqee deddevvecm

841 teeqqlided seavreivdk lqqhqqqqnq qqhhqqlhiq dvvqlaqhsf mpqahsefgn digqemlcda vpmsaaemev sstvitnssn sndssnnisl csstnsltin qmphqasqqp

961 qqnaqsnaqq qrqilvdsng qiignfllqq qrqqqqqqll qqftlqaaaa qqqqqqqqqh qqqqqqqqqa tssnslgktl pvalrngtqq flspnliaqq hqqqqqqqle qhqqqataqq

1081 khqqiqqfal qqaqlhqrql laqaannnll qqqqqqqqnv alpttqakfi akplniismt rpanasptta attantasip sayanvvavt gaqqqqsppv papqqqtvqq qqlanhnsnm

1201 qqlpnvltmk tlppsgvptt iaqqrlqpkm ptgkgrkats nrlppgavnl ersyqicqav iqnspnrenl kaqlrppaai lnqhqptttt apapinpvtl nvstvaatpm snittatgsm

1321 aaavaaappq nvlkqeellv sgavgagalp aglppnvmgv grpgvykvig prmsgfprkk yvqrkpsptt lirhvfspgp ggatataqql qmlqqhhqst tspvpvqnpq qpapeqlihq

1441 ngngqyvlvh ranvgaadnq aprassappm hqnqfvtvqn plhsingipm ggrgrpasvd ttagsgnvia ppisatdalh hhhhemqqqq qhqqpqplgn vgaaanivrr niaagpniay

1561 idgsntnssa valmeagnny ivttnaspta apspinqqpq sqptgtqhqh pllqlhqtge ntppgneata tanncacsln amvicqqcga fchddcigaa klcvacvir

Supplementary Figure 2. The 19 Asx different peptides (grey bars) identified by mass spectrometry in TAP-Calypso purifica-tions all map within an N-terminal 465 amino acid region of Asx (NP_725398).

Supplementary Figure 2 2009-07-08008Scheuermann et al.


SUPPLEMENTARY INFORMATIONdoi: 10.1038/nature08966

Supplementary Figure 3 2009-07-08008 Scheuermann et al.

126

63

45

35

1 2 3 4 5 6 7

*

* * * **

*

# #

+ + + + HA-Asx full length+

+

++++

Flag-SceFlag-Esc

Flag-CalypsoC131S

Flag-Calypso

Flag-Asx full length+

purifiedproteincomplexes(Coomassie)

proteinsexpressedin Sf21 cells

HA-Asx in purifiedcomplexes (western) α-Asx

α-Asx

α-Flag

Sf21-cell extractinput(western)

kDa:

Supplementary Figure 3. Reconstitution of the Calypso-Asx (PR-DUB) complex in vitro.

Upper panel: FLAG-eluates in Coomassie staining. Eluates containing affinity-purifiedFlag-Calypso, Flag-CalypsoC131S, and Flag-Asx are shown in lane 1, 2, and 3. If the Flag-proteins were purified in the presence of HA-Asx containing lysate, HA-Asx co-purified with Flag-Calypso (lane 4) and Flag-CalypsoC131S (lane 5), but not with Flag-Esc, a component of PRC2, (lane 6) and Flag-Sce, a component of PRC1 (lane 7). The respective Flag-proteins are marked with *, HA-Asx is marked with #.

Middle panel: Purified complexes probed with an antibody against Asx (western).Analysis of the elution material (1.5% of total material) shows co-purification of HA-Asx with Flag-Calypso (4) and Flag-CalypsoC131S (5), but not with Flag-Esc (6) and Flag-Sce (7).

Lower panels: Western blots on input material. Input materials (0.05 % of total mate-rial) probed with an Asx-antibody (upper image) or a Flag-antibody (lower image), respectively, show equal amounts of HA-Asx and of the respective Flag-protein in each of the four separate co-purifications.



a

25 kDa 17 kDa 12 kDa

time [min]

H2A-ub1H2A


Calypso �CalypsoAsx2-337

�CalypsoC131S

Asx2-337

2 10 40 2 10 40 2 10 40

un-mod.

H2Aub-modified nucleosomes (Xen.laev.)

1 2 3 4 5 6 7 8 9 10

c

b

2 10 40 2 10 4000

H2A-ub1

H2A

time [min]

- Calypso�Asx2-337

�CalypsoC131S

Asx2-337


un-mod.

H2Aub-modified nucleosomes (Dros.mel.)

1 2 3 4 5 6 7 8

H2A-ub1

H2A

H2B-ub1 25 kDa

17 kDa

H2Bub-modifiednucleosomes (Xen.laev.)

Asx2-337 Asxfull-

25 kDa 25 kDa

17 kDa 17 kDa 12 kDa 12 kDa

30 120 30 120

Calypso ��

H2Aub-modifiednucleosomes (Xen.laev.)

Asxfull-

un-mod.

time [min]

1 2 3 4 5 6

time [min]

7 8 9 10 11 12 kDa

��

30 120 30 120

Supplementary Figure 4. Asx is required as a cofactor for H2A-deubiquitination by Calypso.

a. PR-DUB (Calypso-Asx2-337) rapidly deubiquitinates monoubiquitinated nucleosomal H2A (H2Aub1; lanes 5-7) whereas Calypso alone as well as the mutant complex (CalypsoC131S-Asx2-337) are virtually inactive.

b. PR-DUB (Calypso-Asx2-337) deubiquitinates nucleosomal Drosophila melanogaster (Dros.mel.) H2Aub1 with similar fficiency as nucleosomal Xenopus laevis (Xen. laev.) H2Aub1 (compare to panel a and Fig. 3).

c. PR-DUB complex containing Calypso and Asx1-1668 (Asxfull) deubiquitinates H2Aub1 (lanes 5,6) but not H2B-ub1 (lanes 10,11) in mononucleosomes. Like in the other DUB assays, all reactions contained 30 pmol of Calypso protein. In this set of experiments, longer reaction times were used because Asx1-1668 is present in substoichiometric amounts in PR-DUB complexes with Asx1-1668 (Suppl.Fig. 3, lane 4) as compared to PR-DUB complexes with Asx2-337. Cleavage reactions with Calypso alone (lanes 3,4) or with Calypso-Asx2-337 complex (lanes 8,9) under the same assay conditions served as controls.

15 pmol nucleosomes (corresponding to 30 pmol H2Aub1/H2Bub1) were coincubatedwith 30 pmol enyzme (i.e. Calypso protein) and aliquots of 5 pmol nucleosomes wereanalysed at the indicated time points by western blotting with an antibody against H2Aor H2B, respectively.


e



126

63

45

a b

DNA

GFP

α -Calypso

calypso2 Asx22P4

inset

* *

1 2 3 4

inset


α-tubulin

Calypso

Asx

*

dRing

wt Asx22P4 Asx27J6 calypso2

Supplementary Figure 5. Asx22P4 mutants have reduced levels of PR-DUB subunits Asx and Calypso.

a. Total extracts from 16-18hrs old Drosophila embryos that were wild-type (wt) (lane 1) or homoyzgous for Asx22P4 (lane 2), Asx27J6 (lane 3), or calypso2 (lane 4) were separated on a 4-12% SDS polyacrylamide gel and probed with antibodies against Asx, Calypso, Sce/dRing or α-tubulin as indicated on the right. In Asx22P4 homozygotes, Asx protein signal is diminished to undetectable levels and Calypso protein levels are also strongly reduced (lane 2). The Asx27J6 allele contains a frameshift mutation and encodes a C-terminally truncated Asx protein of 447 amino acids that includes the N-terminal Calypso interaction domain (see Online Methods). In Asx27J6 homozy-gotes, this truncated Asx protein is present and Calypso levels are comparable to those in wt embryos, suggesting that presence of the N-terminal domain of Asx stabilizes the Calypso protein (compare lanes 2 and 3). In calypso2 homozygotes, Calypso protein levels are drastically dimin-ished, the faint residual Calypso signal likely represents maternally deposited wild-type Calypso protein that persisted to these late stages of embryogenesis.

b. Wing imaginal discs from 3rd instar Drosophila larvae with clones of calypso2 (left) or Asx22P4 (right) mutant cells, stained with an antibody against Calypso protein. Mutant clones are marked by the absence of GFP and discs were analysed 96hrs after clone induction. No Calypso protein signal is detected in calypso2 mutant clones and Calypso protein levels are also strongly dimin-ished in Asx22P4 mutant clones, paralleling the observations in embryos.



K63 ub-pentamers [ng]


80 8040 402020

H2A

H2A-ub1

H2A-ub2

1217

25

35

12.5 25 50

H2A [ng]H2A-ub

�-H2A 1:2000 E6C5 (�-H2A-ub) 1:1000

100 200 400 100 200 400


K48 ub-pentamers [ng]H2A-ub

�-ub (P4D1) 1:1000

1217

25

35

45

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

b c

10

17

26

34

43

E6C5 1:1000

�-ub (P4D1) 1:1000

Drosophila embryo nuclear extract [ul] 1 10 1 10

a

Supplementary Figure 6. Assessment of the specificity of commercially available E6C5 antibody.

a. Drosophila embryo nuclear extracts probed with E6C5 antibody (Millipore/ upstate) (left panel) and anti-ubiquitin antibody (right panel).

b. Left panel: 3 pmol recombinant Drosophila mononucleosomes ubiquitinated with RING1B-BMI1 complex (lane 1) and, as reference, different amounts of H2A in unmodi-fied recombinant Drosophila octamers (lanes 2-4), probed with anti-H2A antibody; H2A-ub2 is di-ubiquitinated at H2A-K118 because only H2A-ub1 could be generated in ubiqui-tination assays with Ring1B-Bmi1 complexes and ubiquitin-K all R (i.e. ubiquitin in which all lysine residues had been mutated to arginine) (J.C.S. and J.M., unpublished). Right panel: H2A-ub mononucleosomes (lane 5 contains same amount as lane 1) and different amounts of K63- or K48-linked ubiquitin pentamers (lanes 6-11), probed with E6C5 antibody (Millipore/ upstate).

c. Different amounts of K63- or K48-linked ubiquitin pentamers (lanes 12-17), probed with anti-ubiquitin antibody.




wt

calypso 2

Asx 22P4

(mat +, zyg -)

Sce 1

(mat +, zyg -)

Asx 22P4; Sce 1

(mat -, zyg -)

-

T1 T2 T3 A1 A2 A3 A4 A5 A6 A7 A8

T1 T2 T3 A1 A2 A3 A4 A5 A6 A7 A8

Supplementary Figure 7. Synergy of PR-DUB and Sce/dRing in Polycomb repression.

Ventral view of cuticles of a wild-type embryo (wt) and of embryos that were homozy-gous for calypso2, Asx22P4 or Sce1, or double homozygous for Asx22P4 and Sce1. Thecalypso2 mutant embryo was derived from a female with calypso2 mutant germ cellsand therefore lacked both maternal and zygotic Calypso protein (mat– zyg–). In caseof the other mutants, the homozygous embryos were derived from heterozygousfemales (mat+ zyg–). The calypso2 and Asx22P4 mutant embryos show partial homeo-tic transformation of abdominal segments A5-A7 (arrowheads) into copies of A8(arrow) and abdominal denticles are present in T3 (small arrowhead). Sce1 homozy-gous embryos derived from heterozygous mothers show only mild homeotic trans-formations of A7 to A8 (arrowhead) because these embryos are rescued to a consid-erable extent by maternally deposited wild-type Sce protein. Asx22P4 Sce1 doublehomozygous embryos show very severe homeotic transformations of all body seg-ments (arrowheads) into copies of the A8 segment (arrow) due to widespread mis-expression of Abd-B; this phenotype is comparable to that of Sce1 (mat– zyg–)embryos(1). Repression of HOX genes is thus more severely compromised if both theH2A ubiquitinase Sce and the H2A deubiquitinase PR-DUB are removed.


T1 T2 T3 A1 A2 A3 A4 A5 A6 A7 A8

T3 A1 A2 A3 A4 A5 A6 A7 A8T1 T2

T3 A1 A2A3A4A5A6A7A8T1 T2

(1) Fritsch, C., Beuchle D. and Müller J. Molecular and genetic analysis of the Polycomb group gene Sex combs extra/Ring in Drosophila. Mech. Dev. 120, 949-954 (2003).



α-Abd-B

wt

calypso2

Asx22P4

(mat + zyg -)

(mat - zyg -)

a bAsx clones22P4

halterewing

GFPα-Ubx GFP

α-Ubx

Supplementary Figure 8. Role of PR-DUB in HOX gene regulation.

a. Ventral views of stage 14-16 embryos stained with antibody against the HOX protein Abd-B. (top) wild-type (wt) embryo; (middle) calypso2 homozygous embryo derived from females with calypso2 mutant germ cells and therefore lacking maternal and zygotic calypso+ product (calypsom-z-); (bottom) Asx22P4 homozygous embryo derived from Asx22P4 heterozygous mothers (Asxm+z-). calypso and Asx mutant embryos both show misexpression of Abd-B in the epidermis (arrowheads, compare with wt) but in the central nervous system they also show a partial loss of Abd-B expression (arrows, compare with wt), reminiscent of embryos mutant for the trithorax group genes trithorax or ash1(1). Misexpression of Abd-B in the epidermis is not as widespread as in embryos mutant for other PcG genes(2). In embryos, the requirement for PR-DUB in PcG repression may thus be less pronounced than in larvae.

b. Wing and haltere imaginal disc of 3rd instar larvae with clones of Asx22P4 mutantcells, stained with antibody against Ubx protein. Mutant clones are marked by the absence of GFP and the disc was analyzed 96hrs after clone induction. In the wing imaginal disc, Ubx is misexpressed in Asx mutant clones in the wing pouch (white arrowheads) but remains repressed in clones in the notum and hinge (empty arrow-heads), like in calypso2 mutant clones (Fig.4). In the haltere disc, Ubx expression appears undiminished in Asx mutant clones (arrowhead).


(1) Klymenko, T., and Müller, J. The histone methyltransferases Trithorax and Ash1 prevent transcriptional silencing by Polycomb group proteins. EMBO Rep 5, 373-377 (2004).(2) Soto, M.C., Chou, T.B., and Bender, W. Comparison of germline mosaics of genes in the Polycomb group of Drosophila melanogaster. Genetics 140, 231-243 (1995).



Supplementary Table 1

Sequenced peptides identifying protein bands (shown in Figure 1). Protein

identifications were accepted if the following minimal conditions were met: Presence

of one peptide with a mascot score of at least 45 or presence of two peptides with a

summed score of 40.

Peptides identified Samples identi-fied in silver-stained bands (in-gel digest)

Whole purifi-cation (in-gel digest)

Asx (CG8787)

AAAAAAAAAAAAAAAAAAAAQAQATSSYPSAISPGSK ACLEWR DGKIDLETPDSILASTNLR EASELEQPSSSASGGSPSEAIR ERLSEGEFTPENQLK EVLASIPGFSVKPR HFEPFWGEK IDLETPDSILASTNLR IIVKPIPPEK KPMAPSEEAAVSTAPAPPTR KQAPTTIATINLDDDLDELPSTSK LSASCLNNEFFAR LSEGEFTPENQLK LTTAAQIEQTK LTTAAQIEQTKDGK QAPTTIATINLDDDLDELPSTSK SEPKPPATSQQKPLQQATCDNETELK TTVAVAVADK AGTSQASTMREVLASIPGFSVKPR

++++++

+++++++

+++++++++++++++++++

Calypso (CG8445)

ADEQGESGNGDSQRPDTPTTLLEPSAFT GLAIGNTPELACAHNSHAMPQAR LGIATGEQDIR LLKADEQGESGNGDSQRPDTPTTLLEPSAFT NLDTEIAINEQHLADENDR NLDTEIAINEQHLADENDRR TNQAIVSGTLQK DLQSLLK FNLMAVVPDRR

++

++

++

+++++++++



List of other proteins in the purified material that are not visible as additional bands in figure 1:

Peptides identified

Samples identi-fied in silver-stained bands (in-gel digest)

Whole purification (in-gel digest)

Protein on ecdysone puffs AAAPAAVASPAAAATSADASPSPAK AAAPAAVASPAAAATSADASPSPAKK CIECNKEFATR FFDTEVTAEIHSR IDYDTHLLSAEHLK NQNPPSLLDLPR QTLPISTEEEETR

+

+

+

+++++++

Heat shock cognate 4 ARFEELNADLFR IINEPTAAAIAYGLDKK ISDSDRTTILDKCNETIK LLQDLFNGK LLQDLFNGKELNK MKETAEAYLGK QKELEGVCNPIITK STAGDTHLGGEDFDNR SVIHDIVLVGGSTR TTILDKCNETIK TTPSYVAFTDTER WLDANQLADKEEYEHR DAGTIAGLNVLR KFDDAAVQSDMK LVTHFVQEFK

+

+

+

+

+++

++++++++++++

Tubulin beta-1 chain ALTVPELTQQMFDAK

AVLVDLEPGTMDSVR

INVYYNEASGGK

LHFFMPGFAPLTSR

NMMAACDPR

NSSYFVEWIPNNVK

YLTVAAIFR

+++++++

60S acidic ribosomal protein P0 CFIVGADNVGSK

FAAAASASAAPAAGGATEK GLAVVLMGK GNVGFVFTK TSFFQALSIPTK VVELFDEFPK

++++++

Tubulin alpha-1 chain



AVFVDLEPTVVDEVR

DVNAAIATIK

EDLAALEK

IHFPLVTYAPVISAEK

NLDIERPTYTNLNR

QLFHPEQLITGKEDAANNYAR

++++++

40S Ribosomal protein S3a KDWYDVK LALDSIAKDIEK LIAEDVQDR TVDGYLLR VFEVSLADLQK VVDPFSR

+

+

+

++++++

Elongation factor 1-alpha ALRLPLQDVYK

IGGIGTVPVGR

LPLQDVYK

QTVAVGVIK

VETGVLKPGTVVVFAPANITTEVK

++++

+

++

Ribosomal protein L4 QAYAVSELAGHQTSAESWGTGR GHVIDGVSEFPLVVSDEVQK GYNLPQPK LNPYAEVLK QAVIFLR

+

++++

Ribosomal protein S9 HIDFSLK IGVLDESR IIGEYGLR IPSVFSK LFQGNALLR

+++++

Ubiquitin MQIFVK TITLEVEPSDTIENVK TLSDYNIQK TLSDYNIQKESTLHLVLR

++

++++

Tubulin alpha-4 chain DVNAAVSAIK

ENIAVLER

SIFVDLEPTVIDDVR

TKEELTASGSSASVGHDTSANDAR

++++

Nonmuscle myosin-II heavy chain AAKEELQALSK DLLAKEEGAEEK IANLEEQLENEGKER LNKDIEALER

++++

Ribosomal protein L28 DLTQAALR

GFTAVLK

++



KPFSTEPNNLASVSSYR NNNAFLLK

++

Ribosomal protein L13.e GFTLEELK

GPVLPIKNEQPAVVEFR LILFPINEK TIGIAVDR

++++

Ribosomal protein S6 DIPGLTDTTIPR LITPVVLQR LNVSYPATGCQK LYNLSKEDDVR

+

+

++++

Actin, muscle HQGVMVGMGQKDSYVGDEAQSK SYELPDGQVITIGNER VAPEEHPVLLTEAPLNPK EITALAPSTMK

+++

++++

Ribosomal protein L12 CVGGEVGATSSLAPK HPHDVIDELNEGSIEVPAE IGPLGLSPK

+++

Ribosomal protein S2 EWVPVTK GTGIVSAPVPK LSVVPVR

+++

Ribosomal protein S11 CPWTGDVR

EAIDGTYIDKK QFGVNLNR

+++

Ribosomal protein L6B EVDAALLK TGEADIFAAK VPEHLNDAYFR

+++

Protein lava lamp DAELQDANLVSK

RAKLIER

++

Ribosomal protein L21 GQWVSLK

IGDIVDIK

++

Ribosomal protein L8 SLDFAER

TSGNYATVIAHNQDTK

++

++

Ribosomal protein S8 KFELGRPAANTK

YGKVEQALEDQFTSGR ++

Ribosomal protein L18 AGGEVLTFDQLALR NTLLLQGR

++

Ribosomal protein L27 SLNYNHLMPTR

+



YTAHDISFEK +

Nonmuscle myosin heavy chain VEEQLENEGKER

+

Beta-1 tubulin INVYYNEASGGK

+

H1 histone GGSSLLAIK

+

Mevalonate kinase NTKALVSGVSQR +

Ribosomal protein L10Ab VCILGDQQHCDEAK

+

Ribosomal protein S17 GLQLTQPNTNNFGR

+

Ribosomal protein L15 GLQSIAEER

+

Ribosomal protein DL11 VLEQLTGQQPVFSK

+

Ribosomal protein L23a DYDALDIANK

+

Ribosomal protein S19 IANQIVFK

+

Ribosomal protein L7a NFGIGQNVQPK

+

Ribosomal protein L15 GLQSIAEER

+Ribosomal protein S10b GDVGPGAGEVEFR

+Ribosomal protein L24 AIVGASLAEILAK

+

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