Combinatorial UbiCRest analysis of CTLA4-HA. (A) Line graphs corresponding to the Ub-CTLA4-HA signal shown in Fig. 10 A. (B) Quantification of total ubiquitin released into the supernatant by DUBs relative to USP2 for data represented in Fig. 10 A. Individual data points from two independent, color-coded experiments are shown. Error bars indicate the range. (C and D) Representative western blot showing K29- (C) and K27-linked (D) ubiquitin associated with immunoisolated CTLA4-HA after UbiCRest treatment with indicated DUBs. HeLa S3 Flp-In CTLA4-HA cells were transfected for 72 h with USP8 siRNA prior to lysis. CTLA4-HA was immunoprecipitated using anti-HA magnetic beads and incubated for 1 h at 37°C with the indicated DUBs. (E) Representative western blot of UbiCRest analysis of CTLA4 using a combination of linkage-specific DUBs. HeLa S3 Flp-In CTLA4-HA cells were transfected for 72 h with NT1 or USP8 siRNA prior to lysis. CTLA4-HA was immunoprecipitated using anti-HA magnetic beads and incubated for 1 h at 37°C with the indicated DUBs. Supernatants containing ubiquitin species released by DUBs were collected and analyzed in parallel with CTLA4-HA eluted from the beads. Specific activities of DUBs as reported in the literature are depicted in the key above the blot. Ub-P indicates ability to cleave proximal ubiquitin. Source data are available for this figure: SourceData FS5.