Fig. 3: USP5 deubiquitinates PD-L1.
From: USP5 facilitates non-small cell lung cancer progression through stabilization of PD-L1
A Immunoprecipitates and western blot analysis of cell lysates derived from H1299 cells transfected with HA-Ub, Flag-PD-L1, Myc-USP5 WT/C335A and treated with 5 µM MG132 for 6 h. B H1299 cells with or without USP5 knockdown by siRNA were co-transfected with HA-Ub and Flag-PD-L1, immunoprecipitation was performed using anti-FLAG affinity beads after 5 µM MG132 treatment for 6 h. Poly-ubiquitination of PD-L1 was analyzed by western blot with anti-HA antibodies. C H1299 cells were transfected with or without USP5 siRNA, 72 h after transfection, cells were lysed and subjected to anti-PD-L1 immunoprecipitation (IP), followed by analysis of PD-L1 ubiquitination using immunoblotting with anti-Ub(ubiquitin) antibody. D USP5 deubiquitinates polyubiquitination of PD-L1 in vitro. Flag-PD-L1 and HA-Ub were transfected in HEK293T cells. Poly-ubiquitinated PD-L1 was then immunoprecipitated with anti-Flag M2 beads and incubated with purified USP5-WT/C335A protein as indicated time. Lysates were analyzed by IB with indicated antibodies.
