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. 2021 Apr 6;43(2):429–445. doi: 10.1038/s41401-021-00631-6

Fig. 6. HBXIP acetylates PD-L1 and maintains PD-L1 stability.

Fig. 6

a MCF-7 cells were transfected with pCMV or pCMV-HBXIP vectors, and then treated with CHX (100 μg/mL) for the indicated times. The degradation of PD-L1 protein was analyzed by Western blotting assay. Quantification graph is shown. b MCF-7 and MDA-MB-231 cells were treated with CHX (100 μg/mL) and the indicated concentrations of TSA for 18 h, and endogenous PD-L1 protein levels were evaluated by Western blotting. c Exogenous Flag-PD-L1 was immunoprecipitated with Flag-beads in MCF-7 cells, and then the acetylation level of PD-L1 protein was detected in precipitation samples by Western blotting analysis with anti-acetylated-lysine antibody. d MCF-7 cells were transfected with the indicated vectors. Exogenous Flag-tagged PD-L1 was immunoprecipitated with Flag-beads in MCF-7 cells, and then the acetylation level of PD-L1 was detected by Western blotting analysis with anti-acetylated-lysine antibody. The protein expressions of PD-L1 and HBXIP were examined using anti-Flag or anti-HBXIP antibodies, respectively. e The specificity of anti-acetylated-K270-PD-L1 antibody was detected by dot blot assay. Nitrocellulose membrane was spotted with different amounts of acetylated-K270 peptide (LRKGRMMDVK(AcK)KCGIQDTNSKKQS) or unmodified peptide (LRKGRMMDVKKCGIQDTNSKKQS) and probed with anti-AcK270-PD-L1 (Ac-K270) antibody. f MCF-7 cells were transiently transfected with vector, Flag-PD-L1-WT, Flag-PD-L1-K270R, or Flag-PD-L1-K270Q. Flag-tagged PD-L1 was immunoprecipitated with Flag-beads, and then the acetylated-K270 levels of PD-L1 were tested by Western blotting with anti-AcK270-PD-L1 (Ac-K270) antibody. g MCF-7 cells transiently expressing Flag-PD-L1-WT, Flag-PD-L1-K270R, and Flag-PD-L1-K270Q were treated with CHX (100 μg/mL) for the indicated times, and the protein stabilities of PD-L1 were examined by Western blotting (left). Quantified graph of the PD-L1 protein levels is shown (right). h Flag-PD-L1 vectors accompanied by the indicated plasmids or siRNAs (siRNA of si-CBP#2, si-GCN5#2, si-p300#2, or si-PCAF#2) were transfected into MCF-7 cells. The acetylation and the acetylated-K270 level of PD-L1 protein were tested by Western blotting. i MCF-7 cells were transfected with RFP-HBXIP and/or Flag-PD-L1 vectors. Anti-p300 antibody (red), Flag-PD-L1 (green), and RFP-HBXIP (red) were applied for IF staining. Nuclei were stained with DAPI (blue). Scale bar = 25 μm. Data are presented as mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001, P value was assessed by Student’s t test.