Figure 4.

Siah2 physically interacts with PD-L1 and increases the K63-linked ubiquitination of PD-L1 in CCA. (A), CCA cells were transfected with HA-Siah2 and Flag-PD-L1 for 36 h. Immunoprecipitation was performed with anti-Siah2 and analyzed by immunoblotting. (B), CCA cells were transfected with HA-Siah2 and Flag-PD-L1 for 36 h. Immunoprecipitation was performed with anti-HA and analyzed by immunoblotting. (C), CCA cells with LV-NC/LV-Siah2 were treated with MG132 for 8 h before harvesting, after which cells were harvested in lysis buffer. Supernatants were incubated with the appropriate antibodies overnight. Ubiquitin-modified proteins were analyzed with anti-Ub antibodies. (D), CCA cells with LV-NC/LV-Siah2 were treated with MG132 for 8 h before harvesting, after which cells were harvested in lysis buffer. Supernatants were incubated with the appropriate antibodies overnight. Ubiquitin-modified proteins were analyzed with anti-lysine 48(K48)–linked polyubiquitination or K63-linked polyubiquitination.