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Supplemental Material 1 SUP. FIGURE S1 DAPI NFAT Merge DMSO Ac­5SGlcNAc Figure S1. Inhibition of OGT does not prevent nuclear translocation of NFAT. Jurkat cells !"#$%&'()*+(!!,-.'/012"#..(3'4056'7(+('"+(#"(3'7,"8'9:';<'5=29>/%=45='?+'@<>A'B?+'CD' 8+!E''F(%%!'7(+('"8(-'*%#"(3'?-'#-",2F@GHF@ID2=?#"(3'=?J(+!%,*!'B?+'G:'K,-!E''5B"(+'B,)#",?-L' =(%%!'7(+('#-#%&M(3'$&'=?-B?=#%'K,=+?!=?*&E SUP. FIGURE S2 Labeled Unlabeled Labeled Unlabeled 1h 18h ­Az ­PEG 1h 19h ­Az ­PEG !"#$%"#&'( ­ + ­ + + + !"#$%"#&'( ­ + ­ + + + 110 EWSR1 160 *34+&5 80 &,/ 160 )*+-2$ 160 110 )*+,& 80 60 &,/ 160 110 80 0"1"- RUNX1 110 80 60 60 50 160 SP1 160 110 ELF1 110 80 160 NUP98 110 80 )*+&-. &,/ Figure S2. PEG mass tags allow visualization of O­GlcNAc stoichiometry. 5K PEG mass tags were affixed to O­GlcNAc groups on proteins from control or activated T cells via enzy­ matic labeling with azide and copper­free click chemistry. Proteins were then analyzed for shifts in electrophoretic mobility by immunoblot. For the unlabeled control samples, either the azide (­Az) or PEG (­PEG) reagent was omitted during the labeling procedure. Note that HCFC1 appears as multiple bands because the protein is expressed as a single polypeptide that under­ goes proteolytic processing. UBAP2L appears as two bands in the unlabeled control samples due to alternative splicing. Table S1. Details of 133 higher confidence and 81 lower confidence O-GlcNAc glycoproteins1. Confidence Uniprot ID Symbol Specificity Chi Higher P55265 ADAR 100% 9.11E-04 Higher Q09666 AHNAK 92% 2.36E-16 Higher Q8IWZ3 ANKHD1 100% 1.19E-03 Higher Q8N8A2 ANKRD44 100% 3.39E-02 Higher P50995 ANXA11 100% 2.53E-02 Higher P42331 ARHGAP25 100% 3.39E-02 Higher O14497 ARID1A 100% 8.15E-03 Higher Q6VMQ6 ATF7IP 100% 1.90E-02 Higher Q13315 ATM 100% 6.96E-04 Higher P17655 CAPN2 100% 2.53E-02 Higher Q8IX12 CCAR1 100% 1.08E-04 Higher Q9NXV6 CDKN2AIP 100% 1.43E-02 Higher Q96RK0 CIC 100% 2.53E-02 Higher Q00610 CLTC 100% 3.39E-02 Higher Q99439 CNN2 100% 1.43E-02 Higher A5YKK6 CNOT1 100% 3.39E-02 Higher P57737 CORO7 100% 8.15E-03 Higher Q92793 CREBBP 100% 2.53E-02 Higher P07339 CTSD 100% 4.55E-02 Higher Q7L576 CYFIP1 100% 8.15E-03 Higher Q92841 DDX17 92% 5.55E-03 Higher P23743 DGKA 100% 6.17E-03 Higher O43143 DHX15 100% 1.83E-04 Higher Q5JSL3 DOCK11 100% 4.55E-02 Higher Q8NF50 DOCK8 100% 3.55E-03 Higher Q04637 EIF4G1 100% 4.55E-02 Higher P32519 ELF1 100% 3.55E-03 Higher Q7Z589 EMSY 100% 6.17E-03 Higher Q96L91 EP400 100% 2.03E-07 Higher P07814 EPRS 95% 5.48E-03 Higher Q9BSJ8 ESYT1 100% 1.57E-03 Higher Q9UI08 EVL 90% 2.06E-02 Higher Q01844 EWSR1 95% 9.44E-03 Higher Q01543 FLI1 100% 4.55E-02 Higher O95466 FMNL1 100% 8.15E-03 Higher Q12778 FOXO1 100% 3.39E-02 Higher Q8WXI9 GATAD2B 100% 1.90E-02 Higher Q9HC38 GLOD4 100% 3.39E-02 Higher P63096 GNAI1 94% 2.16E-02 Higher P51610 HCFC1 100% 3.38E-14 Higher P14317 HCLS1 100% 1.43E-02 Higher P31629 HIVEP2 100% 4.55E-02 Higher P01892 HLA-A 94% 1.15E-03 Higher P52597 HNRNPF 100% 8.15E-03 Higher P61978 HNRNPK 94% 2.16E-02 Higher P14866 HNRNPL 100% 8.15E-03 Higher Q9BUJ2 HNRNPUL1 100% 1.08E-02 Higher Q99714 HSD17B10 100% 1.08E-02 Higher Q7Z6Z7 HUWE1 100% 1.90E-02 Higher Q16666 IFI16 90% 2.69E-02 Higher Q14005 IL16 92% 9.37E-03 Higher Q92945 KHSRP 100% 1.08E-02 Higher Q14739 LBR 100% 1.90E-02 Higher P09382 LGALS1 100% 4.55E-02 Higher P02545 LMNA 100% 2.53E-02 Higher P27816 MAP4 100% 1.08E-02 Higher P43243 MATR3 93% 2.85E-02 Higher B0I1T2 MYO1G 100% 4.07E-04 Higher O75376 NCOR1 100% 7.24E-08 Higher P19838 NFKB1 100% 1.90E-02 Higher Q6P4R8 NFRKB 100% 8.15E-03 Higher Q9Y2X3 NOP58 100% 2.05E-03 Higher Q14980 NUMA1 99% 5.61E-15 Higher P49790 NUP153 100% 4.24E-11 Higher P35658 NUP214 100% 7.36E-15 Higher Q7Z3B4 NUP54 100% 6.17E-03 Higher P37198 NUP62 100% 5.32E-04 Higher P52948 NUP98 100% 4.59E-06 Higher Q9BVL2 NUPL1 100% 2.53E-02 Higher Q15365 PCBP1 100% 1.57E-03 Higher Q8WUM4 PDCD6IP 92% 4.99E-02 Higher Q29RF7 PDS5A 100% 4.55E-02 Higher Q8NDX5 PHC3 100% 1.08E-02 Higher Q13492 PICALM 100% 6.17E-03 Higher Q7Z3K3 POGZ 100% 6.96E-04 Higher Q96HA1 POM121 100% 4.87E-05 Higher P14859 POU2F1 100% 1.90E-02 Higher O14974 PPP1R12A 100% 2.03E-07 Higher P48147 PREP 100% 2.05E-03 Higher P78527 PRKDC 99% 1.92E-09 Higher O75400 PRPF40A 100% 4.55E-02 Higher Q96M27 PRRC1 100% 8.15E-03 Higher Q9Y520 PRRC2C 100% 7.13E-09 Higher Q06323 PSME1 100% 8.15E-03 Higher Q9UL46 PSME2 90% 2.06E-02 Higher Q9H3S7 PTPN23 100% 4.55E-02 Higher P08575 PTPRC 98% 1.20E-06 Higher P49792 RANBP2 100% 1.21E-13 Higher Q9NTZ6 RBM12 100% 4.68E-03 Higher Q96PK6 RBM14 100% 2.70E-03 Higher Q5T8P6 RBM26 100% 6.17E-03 Higher Q9P2N5 RBM27 100% 3.39E-02 Higher P46063 RECQL 100% 2.70E-03 Higher Q63HN8 RNF213 100% 1.08E-02 Higher P39023 RPL3 100% 8.15E-03 Higher P62917 RPL8 100% 4.55E-02 Higher P51812 RPS6KA3 100% 8.15E-03 Higher Q01196 RUNX1 100% 4.55E-02 Higher Q9H0E3 SAP130 100% 9.11E-04 Higher Q9UHR5 SAP30BP 100% 4.55E-02 Higher A3KN83 SBNO1 100% 4.55E-02 Higher O15027 SEC16A 100% 1.43E-02 Higher Q9Y6Y8 SEC23IP 100% 6.96E-04 Higher O95487 SEC24B 100% 4.68E-03 Higher P53992 SEC24C 100% 4.68E-03 Higher O94979 SEC31A 100% 2.70E-03 Higher P30740 SERPINB1 100% 4.68E-03 Higher P50453 SERPINB9 100% 2.05E-03 Higher Q15637 SF1 100% 9.11E-04 Higher Q15459 SF3A1 100% 4.68E-03 Higher P23246 SFPQ 100% 1.08E-02 Higher Q08AF3 SLFN5 100% 2.53E-02 Higher Q8TAQ2 SMARCC2 100% 1.90E-02 Higher P08047 SP1 100% 3.39E-02 Higher Q01082 SPTBN1 100% 1.57E-03 Higher Q6ZRS2 SRCAP 100% 1.08E-02 Higher Q9UQ35 SRRM2 94% 2.16E-02 Higher P42224 STAT1 100% 2.70E-03 Higher Q03518 TAP1 100% 6.17E-03 Higher Q13428 TCOF1 95% 4.18E-03 Higher P12270 TPR 100% 3.55E-03 Higher Q14157 UBAP2L 100% 2.39E-04 Higher P45880 VDAC2 100% 2.70E-03 Higher Q9Y277 VDAC3 96% 2.44E-03 Higher Q14119 VEZF1 100% 5.32E-04 Higher Q9H4A3 WNK1 100% 7.13E-09 Higher P12956 XRCC6 100% 1.90E-02 Higher P49750 YLPM1 100% 3.39E-02 Higher P43403 ZAP70 100% 6.96E-04 Higher Q7Z2W4 ZC3HAV1 100% 3.39E-02 Higher Q96KR1 ZFR 100% 1.40E-04 Higher O43670 ZNF207 100% 1.08E-02 Higher Q15942 ZYX 100% 2.70E-03 Lower P49588 AARS 100% - Lower Q15027 ACAP1 100% - Lower P52594 AGFG1 100% - Lower P23526 AHCY 100% - Lower Q10567 AP1B1 100% - Lower Q92888 ARHGEF1 92% - Lower Q86VP6 CAND1 100% - Lower P20810 CAST 100% - Lower P49368 CCT3 100% - Lower P06729 CD2 100% - Lower P16070 CD44 100% - Lower Q9NYV4 CDK12 100% - Lower Q14019 COTL1 92% - Lower O75131 CPNE3 100% - Lower P41240 CSK 100% - Lower Q96F07 CYFIP2 100% - Lower Q9GZR7 DDX24 100% - Lower P35659 DEK 100% - Lower Q08211 DHX9 92% - Lower O60496 DOK2 100% - Lower Q9HC35 EML4 100% - Lower Q9NUQ9 FAM49B 100% - Lower P85037 FOXK1 100% - Lower Q14697 GANAB 100% - Lower P09211 GSTP1 100% - Lower P40939 HADHA 100% - Lower P51858 HDGF 100% - Lower Q04826 HLA-B 92% - Lower Q12906 ILF3 90% - Lower Q92835 INPP5D 100% - Lower P20701 ITGAL 100% - Lower Q7LBC6 KDM3B 100% - Lower Q5XKE5 KRT79 100% - Lower P06239 LCK 100% - Lower P47929 LGALS7 100% - Lower Q9UIQ6 LNPEP 100% - Lower P33241 LSP1 90% - Lower Q9NR56 MBNL1 100% - Lower O60318 MCM3AP 100% - Lower O94776 MTA2 100% - Lower P14649 MYL6B 100% - Lower Q15233 NONO 92% - Lower Q16656 NRF1 100% - Lower P22061 PCMT1 100% - Lower O00329 PIK3CD 100% - Lower Q14651 PLS1 100% - Lower P29590 PML 100% - Lower Q6NYC8 PPP1R18 100% - Lower P30153 PPP2R1A 100% - Lower Q9UHX1 PUF60 100% - Lower Q00577 PURA 100% - Lower P11216 PYGB 100% - Lower Q9P258 RCC2 100% - Lower Q04206 RELA 100% - Lower P27635 RPL10 100% - Lower P46779 RPL28 90% - Lower P04843 RPN1 100% - Lower Q5VT52 RPRD2 100% - Lower P25398 RPS12 100% - Lower Q14141 SEPT6 92% - Lower Q9UHD8 SEPT9 91% - Lower P29508 SERPINB3 100% - Lower P31947 SFN 100% - Lower P05141 SLC25A5 100% - Lower P08195 SLC3A2 100% - Lower Q92922 SMARCC1 100% - Lower A6NHR9 SMCHD1 100% - Lower Q02447 SP3 100% - Lower P37108 SRP14 100% - Lower P05455 SSB 100% - Lower P40763 STAT3 100% - Lower Q9UH99 SUN2 90% - Lower O15260 SURF4 100% - Lower P49848 TAF6 100% - Lower Q9NXG2 THUMPD1 100% - Lower O14773 TPP1 100% - Lower Q9UHD9 UBQLN2 100% - Lower P45974 USP5 100% - Lower Q93009 USP7 100% - Lower P21796 VDAC1 100% - Lower P27348 YWHAQ 100% - 1. Proteins are classified as higher confidence hits (at least 8 total sequenced peptides, at least 2 unique sequenced peptides, a chi-square p-value of less than 0.05, and at least 90% of total peptides originating from the labeled conditions) or lower confidence hits (at least 4 total sequenced peptides, at least 2 unique sequenced peptides, and at least 90% of total peptides originating from the labeled conditions). Proteins are listed by their UniProt ID, symbol, and percent of total spectral counts in the labeled conditions (specificity). To provide an objective measure of whether the protein is significantly enriched in the labeled conditions, we performed chi-square tests for the higher confidence proteins to assess whether the observed total spectral counts from the labeled and unlabeled conditions significantly differed from the expected values (2/3 of total counts from labeled conditions and 1/3 from unlabeled).
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