Fig. 2: TMUB1 stabilizes PD-L1 by antagonizing its polyubiquitination by the E3 ligase HUWE1. | Nature Communications

Fig. 2: TMUB1 stabilizes PD-L1 by antagonizing its polyubiquitination by the E3 ligase HUWE1.

From: Promoting anti-tumor immunity by targeting TMUB1 to modulate PD-L1 polyubiquitination and glycosylation

Fig. 2: TMUB1 stabilizes PD-L1 by antagonizing its polyubiquitination by the E3 ligase HUWE1.The alternative text for this image may have been generated using AI.

a, b Immunoblots of PD-L1 in MDA-MB-231 cells with stable overexpression of Tag-free TMUB1 (a), TMUB1-knockdown (b) or control following treatment with 20 µg/mL cycloheximide (CHX) for the indicated time points. c Immunoblots of PD-L1 in TMUB1-knockdown or control MDA-MB-231 cells following treatment with 10 µM of MG132 or 50 µM of CQ for 6 h. Data are presented as mean ± SEM; n = 4. One-way ANOVA followed by Tukey’s test. Co-IP analysis of the interaction between endogenous ubiquitin and PD-L1 in stable Tag-free TMUB1-overexpression (d), TMUB1-knockdown (e) or control MDA-MB-231 cells treated with 10 µM of MG132 for 6 h. f The MS analysis to explore different PD-L1-binding proteins in Fig.1d. Numbers represent the peptide-spectrum matches of different proteins detected by mass spectrometry in the indicated groupings. The representative candidates are listed. TMUB1 and HUWE1 were identified. g Co-IP analysis of the interaction between endogenous PD-L1 and HUWE1-HA in MDA-MB-231 cells treated with 10 µM of MG132 for 6 h. h, i Immunoblots of PD-L1 in TMUB1-knockdown (h), Tag-free TMUB1-overexpression (i) or control MDA-MB-231 cells transfected with HA-empty vector or HA-HUWE1. j TUBE-pull down assay of the interaction between PD-L1-Flag and endogenous ubiquitin in stable TMUB1-His or empty-vector overexpression MDA-MB-231 cells transfected with HA-HUWE1 or HA-empty vector. k The working model of TMUB1 antagonizing HUWE1-mediated PD-L1 ubiquitination and enhancing PD-L1 stability.

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