USP8 depletion dramatically enhances CTLA4 ubiquitylation. (A) Representative western blots of TUBES pulldown of ubiquitylated CTLA4. HeLa S3 Flp-In CTLA4-HA and A2058 cells were transfected for 72 h with non-targeting (NT1), USP8, and AMSH siRNA prior to lysis. Lysates were subjected to TUBES-pulldown (IB: Immunoblot). (B) Quantification of ubiquitylated CTLA4-HA isolated by TUBES pulldown for data represented in A. Ubiquitylated CTLA4-HA was normalized to total ubiquitin pulled down. Individual data points from two independent, color-coded experiments are shown. Error bars indicate the range. (C) Representative western blots of ubiquitylated CTLA4-HA immunoprecipitated under denaturing conditions. HeLa S3 Flp-In parental (Par) or CTLA4-HA (HA) cells were transfected for 72 h with non-targeting (NT1), USP8, and AMSH siRNA. Cells were lysed in denaturing SDS lysis buffer and lysates were subjected to immunoprecipitation (IP) with anti-HA coupled magnetic beads. *Antibody heavy chain; ** antibody light chain. (D) Quantification of CTLA4-HA ubiquitylation relative to NT1 for data represented in C. Ubiquitylated CTLA4-HA was normalized to total immunoprecipitated CTLA4-HA. Individual data points from two independent, color-coded experiments are shown. Error bars indicate the range. (E) Representative western blots of TUBES pulldown of ubiquitylated CTLA4. Lysates from USP8 fl/fl (Control, n = 3) and USP8 deleted (∆USP8, n = 3) activated T cells derived from individual mice were either analyzed directly by SDS-PAGE and western blot (left blot), or first subjected to a TUBES pulldown prior to analysis alongside input samples. (F) Quantification of ubiquitylated CTLA4 enriched by TUBES pulldown for data represented in E. Ubiquitylated CTLA4 was ratioed to total CTLA4 levels. Individual data points from six individual mice are shown. Error bars indicate SD. Source data are available for this figure: SourceData F4.
Figure 4.

USP8 depletion dramatically enhances CTLA4 ubiquitylation. (A) Representative western blots of TUBES pulldown of ubiquitylated CTLA4. HeLa S3 Flp-In CTLA4-HA and A2058 cells were transfected for 72 h with non-targeting (NT1), USP8, and AMSH siRNA prior to lysis. Lysates were subjected to TUBES-pulldown (IB: Immunoblot). (B) Quantification of ubiquitylated CTLA4-HA isolated by TUBES pulldown for data represented in A. Ubiquitylated CTLA4-HA was normalized to total ubiquitin pulled down. Individual data points from two independent, color-coded experiments are shown. Error bars indicate the range. (C) Representative western blots of ubiquitylated CTLA4-HA immunoprecipitated under denaturing conditions. HeLa S3 Flp-In parental (Par) or CTLA4-HA (HA) cells were transfected for 72 h with non-targeting (NT1), USP8, and AMSH siRNA. Cells were lysed in denaturing SDS lysis buffer and lysates were subjected to immunoprecipitation (IP) with anti-HA coupled magnetic beads. *Antibody heavy chain; ** antibody light chain. (D) Quantification of CTLA4-HA ubiquitylation relative to NT1 for data represented in C. Ubiquitylated CTLA4-HA was normalized to total immunoprecipitated CTLA4-HA. Individual data points from two independent, color-coded experiments are shown. Error bars indicate the range. (E) Representative western blots of TUBES pulldown of ubiquitylated CTLA4. Lysates from USP8 fl/fl (Control, n = 3) and USP8 deleted (∆USP8, n = 3) activated T cells derived from individual mice were either analyzed directly by SDS-PAGE and western blot (left blot), or first subjected to a TUBES pulldown prior to analysis alongside input samples. (F) Quantification of ubiquitylated CTLA4 enriched by TUBES pulldown for data represented in E. Ubiquitylated CTLA4 was ratioed to total CTLA4 levels. Individual data points from six individual mice are shown. Error bars indicate SD. Source data are available for this figure: SourceData F4.

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