MG132. GRXS17:V5-His MG132 GRXS17:HA. RuBisCo. Supplemental Data. Walton et al. (2015). Plant Cell /tpc
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1 MG132 GRXS17:V5-His MG132 GRXS17:HA RuBisCo Supplemental Figure 1. Degradation of GRXS17 by the 26S proteasome. (A) Cell-free degradation assay. Recombinant protein GRXS17:V5-HIS was incubated with total protein extract prepared from wild-type Arabidopsis seedlings in the presence or absence of MG132 for the indicated time. (B) Total proteins extracted from 35S:GRXS17:HA seedlings and incubated in the presence or absence of MG132. Samples were harvested at the indicated times. Coomassie Blue-stained ribulose-1,5- bisphosphate carboxylase/oxygenase (RuBisCo) was used as loading control. Proteins were detected with anti-v5 (1/2000) or anti-ha (1/1000) antibodies with their respective horseradish peroxidase (HRP)- conjugated secondary antibody.
2 Supplemental Figure 2. The Ubiquitination Site website page. In a query box, one or multiple AT numbers can be entered, returning the ubiquitination sites on corresponding proteins and information concerning the splice variant, modified sequence, sequence window, and the site position within the protein. The MAXQUANT score and delta score are provided as well. Through its link to PLAZA, access can be gained to ample information concerning the protein(s) in question. Digestion - LysC Tryp Acetylation - A - A - A Supplemental Figure 3. Acetylation efficiency testing. Silver-stained SDS-PAGE displaying the resistance of acetylated proteins to cleavage by endolysc. The used proteolytic enzyme [none (-), endolysc (LysC), or trypsin (Tryp)] and acetylation status [mock (-) or acetylated (A)] are indicated above the panels.
3 Supplemental Table 1. Identified ubiquitination sites on transcription factors and UPS members. Type Entry AGI code Description Modified peptide sequence Transcription factors bzip MYB WRKY bhlh TCP AT1G43700 Transcription factor VIP1 (Basic leucine zipper 51) (AtbZIP51) AT3G17609 Transcription factor HY5-like protein HYH (bzip transcription factor AtbZip64) AT5G11260 Transcription factor HY5 (Protein LONG HYPOCOTYL 5) AT1G72650 Myb family transcription factor TRFL6 AT2G04880 WRKY transcription factor 1 (Transcription factor ZAP1) AT4G26640 WRKY20 AT4G22070 WRKY31 AT4G30935 WRKY32 AT2G38470 WRKY33 AT3G01970 WRKY45 AT5G49520 WRKY48 AT4G36930 Transcription factor SPATULA (SPT) (Basic helix-loop-helix protein 24) AT4G02590 Transcription factor UNE12 (Basic helix-loophelix protein 59, bhlh 59) AT1G58100 TCP8 AT1G72010 TCP22 Linked to hormones ABA AT1G45249 Abscisic acid responsive elements-binding factor 2 ABF2 AT3G56850 ABSCISIC ACID-INSENSITIVE 5-like protein 2 DPBF3 (ABA-responsive element-binding protein 3) Auxin AT1G19220 Auxin response factor 19 ARF19 (Auxinresponsive protein IAA22) AT5G62000 Auxin response factor 2 ARF2 (ARF1-binding protein, ARF1-BP) Ethylene AT5G13330 Ethylene-responsive transcription factor ERF113 Brassinosteroids AT5G07310 Ethylene-responsive transcription factor ERF115 _GTSELNTENK(Ubi)HLKMR LAELALLDPK(Ubi)R KK(Ubi)VYVSDLESR VPEFGGEAVGK(Ubi)ETSGR TPAEK(Ubi)ENK(Ubi)R DSVEK(Ubi)SASR LSEK(Ubi)SEVR E(Ubi)VMEDGYNWR KK(Ubi)GGNIELSPVER SDVFTAVSKEK(Ubi)TSGSSVQTLR KYGQK(Ubi)VVR NSSQDHLLAQESK(Ubi)AEGR DEEK(Ubi)SLGADMEDLHDETVR ETLGK(Ubi)DQVQGVR K(Ubi)TSFSPR KYGQKQVK(Ubi)GSENPR LTEFHGVDNSAQPTTSSEEK(Ubi)PR SDTINTQTNEENK(Ubi)K AAEVHNLSEK(Ubi)R ALQELVPTVNK(Ubi)TDR STPPEDSTLATTSSTATATTTK(Ubi)R SVDLSK(Ubi)ENDDR EDYFK(ac)EPSSAAEPSESSQK(Ubi)ASQFQEQELAQGR KSGTVEK(Ubi)VVER VASGEVVEK(Ubi)TVER TYTKVQK(Ubi)R ASSEVSMK(Ubi)GNR DPKK(Ubi)AAR EEEEK(Ubi)NYGYNYYNYPR ANSGNYGK(Ubi)R_
4 Ubiquitin conjugation E1 AT5G06460 Ubiquitin-activating enzyme E1 2 UBA2 E2 AT5G50870 Ubiquitin-conjugating enzyme E2 27 UBC27 (Ubiquitin carrier protein 27) AT1G70660 Ubiquitin-conjugating enzyme E2 variant 1B UEV1B (Ubc enzyme variant 1B) AT2G36060 Ubiquitin-conjugating enzyme E2 variant 1C UEV1C (Ubc enzyme variant 1C) E3 UBOX ASK F-box 26S AT1G01680 U-box domain-containing protein 54 PUB54 AT5G05230 U-box domain-containing protein 62 PUB62 AT2G45950 SKP1-like protein 20 ASK20 AT3G61415 SKP1-like protein 21 ASK21 AT1G20140 SKP1-like protein 4 ASK4 _LEDVNSK(Ubi)LLR VCPK(Ubi)SDNLTR GSEEEK(Ubi)VVVPR GEK(Ubi)GIGDGTVSYGMDDGDDIYMR K(Ubi)LVQPPEGTFF KETIEK(Ubi)SKSNESDEDPR KETIEK(Ubi)SKSNESDEDPR VGEQDPK(Ubi)TR IIEGK(Ubi)NPEEIR LK(Ubi)NVEVEEHVDER IIEGK(Ubi)TPEEIR GK(Ubi)TPEQMR_ AT2G16365 F-box protein _K(Ubi)NESSAETNTLEMDR LQSLESSK(Ubi)DTQEDGPR_ At1g47765 Putative F-box protein _FELK(Ubi)EIADDQAR_ AT4G39756 Putative F-box/kelch-repeat protein _DIK(Ubi)GLATLNR_ AT5G S protease regulatory subunit 10B homolog A RPT4A AT3G S protease regulatory subunit 6A homolog A RPT5A AT1G S protease regulatory subunit 7 homolog A RPT1A AT5G S proteasome non-atpase regulatory subunit 12 homolog B RPN5B AT4G S proteasome regulatory subunit 4 homolog A RPT2A Deubiquitinating enzymes AT3G11910 Ubiquitin carboxyl-terminal hydrolase 13 UBP13 AT1G51710 Ubiquitin carboxyl-terminal hydrolase 6 UBP6 AT4G17510 Ubiquitin carboxyl-terminal hydrolase UCH3 _K(Ubi)IEIPLPNEQSR SKVDKEK(Ubi)LTSGTR K(Ubi)IEFPHPTEEAR DIEDEIRDEK(Ubi)NPR LLNEEK(Ubi)QMR LKPQEEK(Ubi)AEEDR AEEIPEEEK(Ubi)NIGPNDR KK(Ubi)LEAPR ATASESSSSK(Ubi)R_
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