condition. Left panel, the HCT-116 cells were lysed with RIPA buffer containing 0.1%
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1 FIGURE LEGENDS Supplementary Fig 1 (A) sumoylation pattern detected under denaturing condition. Left panel, the HCT-116 cells were lysed with RIPA buffer containing 0.1% SDS in the presence and absence of NEM. Right panel, the HCT-116 cells were lysed with RIPA buffer containing 1% SDS in the presence and absence of NEM. The lysate were then heated at 100 C for 5 minutes. After cooling down on ice, the samples were diluted to bring the final SDS concentration to 0.1% and then subjected to IP and subsequent IB. (B) Antibody against SUMO-1 does not cross reacts with ubiquitin. The whole lysate of HCT-116 cells were used for IP with ant-sumo-1 or rabbit IgG. After blot with anti-sumo-1 (left panel), the identical blot was stripped and reblotted with anti-ubiquitin. Supplementary Fig 2 Single or double mutation of SUMO sites does not inhibit NIK-induced processing. The HCT-116 cells were co-transfected with or SUMO mutants along with or without NIK. 24 hour after transfection, the cells were lysed for IB with anti- N. Supplementary Fig 3 SUMO modification on stimuli-induced phosphorylation. (A) Single or double mutation of SUMO sites does not affect NIK-induced phosphorylation at SS866/867 and processing. (B) The mouse M12 CD40 B cells were stimulated with anti-murine CD40 (2 μg/ml) in the presence of MG132 (10 μm) for the indicated time. Whole cell lysate were used for IP with anti- N and IB with anti- 1
2 SUMO-1 (top panel) or anti-phospho- at S866/870 (middle panel). The identical blot used for evaluating SUMO-1 conjugation (top panel) was stripped and reblotted with anti- N to show the pattern of modified (bottom panel). Supplementary Fig 4 SUMO modification does not affect cellular distribution and interaction with IKKα. (A) The HCT-116 cells were transfected with and mutant. The cytosolic fractions (CE) and nuclear fractions (NE) were separated and used for IB. (B) The HCT-116 cells were transfected with and mutant along with or without NIK. 48 hour after transfection, the transfected cells were harvested and the whole cell lysate were used for IP and IB. Supplementary Fig 5 The HEK 293 cells were transfected with or mutant along with NIK. 24 hour after transfection, the cells were harvested and cytosolic fractions (CE) and nuclear fractions (NE) were separated and used for IP and IB. Supplementary Fig 6 (A) The -/- NIH 3T3 cells were transfected with and mutant along with NIK. The cells were harvested 48 hour after transfection. The CE and NE fractions were used for the IP with anti-c-myc and subsequent IB with RelA, RelB and N. (B) The total mrnas and the whole cell lysate were used for evaluating Skp2 mrna and Skp2 protein level respectively. To minimize the interference resulting from unequal mrna input, the probes for β-actin and the probes for Skp2 were added to the same RT-PCR reaction tube. 2
3 Supplementary Figure 1 A B IP: IgG IP: N IP: IgG IP: N IP: IgG IP: SUMO-1 IP: IgG IP: SUMO-1 NEM IB: SUMO KDa 150 KDa 100 KDa SUMO- RIPA buffer (0.1% SDS) RIPA buffer (1% SDS) & pre-boiling IB: SUMO-1 IB: Ubiquitin
4 Supplementary Figure 2 NIK IB: N K863R K689R K90R KK90/689RR
5 Supplementary Figure 3 A K863R K689R K90R K863R K689R K90R K298 KK298/689RR KKK90/298/689RRR K298 KK298/689RR KKK90/298/689RRR IB: p- p- IB: N -NIK + NIK -NIK + NIK B IP: N CD40 (time) h 2h 250KDa 150KDa 100KDa SUMOp- SUMO IB: SUMO-1 250KDa 150KDa 100KDa IB: p- 250KDa 150KDa 100KDa Modified IB: N M12 CD40 B cell
6 Supplementary Figure 4 A NIK IP: N IB: N B NIK IP: N IB: IKKα IKKα IB: IKKα IKKα Input CE NE CE NE
7 Supplementary Figure 5 NIK CE NE IB: RelA IB: RelB IB: N IB: Sp1 RelA RelB Sp1 HEK 293
8 Supplementary Figure 6 A IB: RelA IB: RelB IB: N CE NIK NE RelA RelB NIH 3T3 -/- B NIK Skp2 mrna Actin mrna Skp2 protein GAPDH protein NIH 3T3 -/- NIH 3T3 -/-
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doi: 1.138/nature89 IFN- (ng ml ) 5 4 3 1 Splenocytes NS IFN- (ng ml ) 6 4 Lymph node cells NS Nfkbiz / Nfkbiz / Nfkbiz / Nfkbiz / IL- (ng ml ) 3 1 Splenocytes IL- (ng ml ) 1 8 6 4 *** ** Lymph node cells
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