Fig. 3.

FBXO22 mediates ubiquitination and degradation of PD-L1. (A and B) PD-L1 levels were determined by Western analysis after treatment with MG132 (30 µM) for 6 h in A549 cells overexpressing FBXO22 (A) or transfected with the empty vector control (B). (C) Endogenous ubiquitination of PD-L1 was determined in Calu1 cells overexpressing FBXO22 (FBX) or the empty vector (Vec) after treatment with MG132 (30 µM for 6 h). Whole-cell lysates of MG132-treated cells were used for immunoprecipitation of PD-L1 and the levels of ubiquitination and PD-L1 were measured by the Western method. (D and E) Ubiquitination of PD-L1 in A549 cells, with or without manipulation of FBXO22 (overexpression in C; KO in D). The cells were stimulated with interferon-γ (1 ng/mL) for 16 h, followed by MG132 (30 µM for 6 h). Immunoprecipitation was followed by Western analysis.