Fig. 3: TMUB1 protects PD-L1 from HUWE1-mediated ER-associated degradation.

a, b Co-IP analysis and immunoblots for PD-L1-HA and TMUB1-Flag (a) or HUWE1-Flag (b) in the HEK-293T cells treated with 5 μg/ml Tunicamycin for 12 h or not or 10 µM of MG132 for 6 h. Triangle: Glycosylated PD-L1, Circle: Non-glycosylated PD-L1. c, d Endoplasmic reticulum localization of TMUB1 (c) and HUWE1 (d) was detected using immunofluorescence staining (left), Calnexin was stained to characterize the endoplasmic reticulum. Line scan of the relative fluorescence intensity of the signal (dotted line; left), demonstrating peak overlapping (right). Scale bar: 10 µm. e Analysis of the subcellular localization of PD-L1, TMUB1, and HUWE1 in the fraction of MDA-MB-231 cells isolated by differential centrifugation. f Co-IP analysis for PD-L1 and TMUB1/HUWE1 in the ER fraction of MDA-MB-231 cells. g Co-IP analysis for the interaction of the different truncations of PD-L1-HA with TMUB1-Flag in HEK-293T cells. h Co-IP analysis for the interaction of the different truncations of PD-L1-Flag with HUWE1-HA in HEK-293T cells. i Schematic diagram for PD-L1 displaying the position of the Extracellular Domain (ECD) and the Transmembrane and Cytoplasmic Domain (T + C). j Co-IP analysis of the interaction between endogenous HUWE1 and PD-L1 within MDA-MB-231 cells with TMUB1 overexpression or not. IgG was used as the negative control. k Immunoblots of PD-L1 in TMUB1-knockdown or control MDA-MB-231 cells following treatment with 10 µM of Eer I for 12 h. Cell lysates were treated with PNGase F. Triangle: Glycosylated PD-L1, Circle: Non-glycosylated PD-L1. l Immunoblots of PD-L1 in TMUB1-knockdown or control MDA-MB-231 cells following treatment with 10 µM of MG132 and 50 µM of CQ for 6 h. Cell lysates were treated with PNGase F. Triangle: Glycosylated PD-L1, Circle: Non-glycosylated PD-L1. m Volcano plot represented the gene correlation with TMUB1 in breast cancer, p-value < 0.01 and absolute correlation coefficient >0.3 or <−0.3 by using Proteome data of CPTAC - Breast invasive carcinoma prospective cohort from Linkedomics. Two-sided t test. n Co-IP analysis for the interaction between endogenous PD-L1 and endogenous STT3A within MDA-MB-231 cells with TMUB1-Overexpression or not. IgG was used as the negative control.