Fig. 1
PD-L1 is modified by UBL3. (a) Co-immunoprecipitation (IP) analysis of using MDA-MB-231 cell lysates expressing Flag-tagged PD-L1 (PD-L1-Flag) and biotinylated-tagged UBL3 (Biotin-UBL3). (b) Co-IP was performed using cell lysates expressing PD-L1-Flag or PD-L1 mutants-Flag (C250A, C272A, C250/272A) along with Biotin-UBL3 in MDA-MB-231 cells. IB analysis was conducted using the indicated antibodies. (c) the relative intensity of UBL3 modification (Flag/SA-HRP) calculated as the ratio of UBL3-modified PD-L1 signals (> 70 kDa) to input SA-HRP signals, normalized to the mean of cells transfected with Biotin-UBL3 and wild-type PD-L1. Data are presented as mean ± s.e.m., with dots representing individual experiments. One-way ANOVA with Tukey’s multiple comparisons test. ns, not significant, *P < 0.05, **P < 0.001, ***P < 0.0005, PD-L1 vs. C250A, P = 0.1708; PD-L1 vs. C272A, P = 0.0009; PD-L1 vs. C250/272A, P = 0.0002; C250A vs. C272A, P = 0.0135; C272A vs. C250/272A, P = 0.5847; TMD, transmembrane domain; Flag, Flag-tag; βME-, without 2-mercaptoethanol; βME+, with 2-mercaptoethanol; SA-HRP, streptavidin-horseradish peroxidase.
