Extended Data Fig. 6: Succinylation mainly occurred in PD-L1 K129 to control its stability.
From: Alterations in PD-L1 succinylation shape anti-tumor immune responses in melanoma
a, Endogenous succinylated proteins were immunoprecipitated using a pan-succinylated lysine antibody, and PD-L1 protein was detected in SK-MEL-28, H460, MDA-MB-231, and HCT-116 cells by immunoblotting. b, IP/MS analysis identified the succinylation site on PD-L1, with a secondary mass spectrometry profile showing succinylation at the K129 site. c, Western blot analysis of whole-cell lysates from A375 cells stably expressing wild-type (WT) or mutant PD-L1 (K136E, K136R). Cells were treated with cycloheximide (CHX) for the indicated times (n = 3 independent experiments). PD-L1 protein levels were quantified by ImageJ. d, PD-L1 WT or mutants were transfected into SK-MEL-28 cells and treated with tunicamycin for 24 hours. PD-L1 expression was analyzed by immunoblotting (n = 3 independent experiments). e, PD-L1 WT or mutants were transfected into SK-MEL-28 cells for 24 hours, followed by 24-hour treatment with tunicamycin. Protein was harvested for PD-L1 detection (n = 3 independent experiments). f, g, Representative immunofluorescence images showing the localization of PD-L1 WT and mutants in A375 cells (n = 3 independent experiments), with markers for the Endoplasmic Reticulum (GRP94) (f) and Golgi apparatus (TGN46) (g). Scale bar, 7.5 μm.
