Fig. 3: GSK3α-mediated phosphorylation of PD-L1 promotes PD-L1 degradation.
From: ARIH1 signaling promotes anti-tumor immunity by targeting PD-L1 for proteasomal degradation
a Immunoblots of PD-L1 (anti-Flag) in HEK293T cells transfected with Flag-PD-L1 (WT) or Flag-PD-L1 (mutants) following treatment with 25 ng/mL EGF and/or 10 μM ES-072 for 48 h, 2SA represents T180A/S184A. b HEK293T cells were transfected with Flag-PD-L1 and treated with ES-072 (10 μM) for 48 h. Proteins that co-immunoprecipitated (Co-IP) with PD-L1 were analyzed by mass spectrometry. Kinases and kinase-related proteins are shown in heatmap. c Co-IP analysis for the interaction of GSK3α/GSK3β and PD-L1 in HEK293T cells transfected with Flag-PD-L1 and treated with or without 10 μM ES-072 for 12 h, Flag-tagged empty vector (Flag-EV) was transfected as a negative control. d Proximity ligation assay (PLA) analysis for the interaction of GSK3α and PD-L1 in HEK293T cells treated as c. PLA signals are shown in red and the nuclei in blue; scale bar, 20 μm. Quantification for the mean area (MA) of PD-L1/GSK3α PLA speckles is indicated by scattergram. Data represent means ± SEM, n = 50, ****P < 0.0001. e Immunoblots of p-GSK3α, GSK3α, p-GSK3β, GSK3β, p-AKT, AKT, p-EGFR, and EGFR in H1975 cells treated with 10 μM ES-072 for indicated times. f In vitro GSK3α kinase assay was performed in the presence or absence of ATP. The phosphorylation of PD-L1 peptides (PD-L1-WT/2SA) was detected by dot blot with anti-p-PD-L1 (Ser279) antibody, p-PD-L1 peptides were synthesized as a positive control. g In vitro GSK3α kinase assay was performed in HEK293T cells transfected with Flag-PD-L1 and GSK3α-HA. Total cell lysates were immunoprecipitated with anti-Flag or anti-HA. The phosphorylation of PD-L1 by GSK3α was detected using an anti-p-PD-L1 (Ser279) antibody. h Immunoblots of p-PD-L1, PD-L1, and GSK3α in H1975 cells transfected with GSK3α-siRNAs. i Immunoblots of PD-L1 and GSK3α in U937 cells transfected with GSK3α-siRNAs and treated with or without 10 μM ES-072 for 24 h. j Immunoblots of PD-L1 (anti-Flag) and GSK3α in HEK293T cells transfected with Flag-PD-L1 following treatment with GSK3α-siRNA and treated with or without 10 μM ES-072 for 24 h. Source data are provided as a Source Data file.
