Fig. 3: Abnormal N-glycosylation of B7H3 at N91/N309 and N104/N322 induces its ER-associated degradation.
From: Targeting site-specific N-glycosylated B7H3 induces potent antitumor immunity
a IP-MS analysis showing candidates with increased binding to 8NQ or N91/104/309/322Q B7H3 compared to WT or N91/104/309/322Q B7H3. b The indicated cell lines were treated with MG132, NMS-873 (2 μM) or CB-5083 (5 μM) in the presence of CHX at indicated intervals. c The indicated cell lines were treated with NMS-873 or CB-5083. Immunoprecipitation analysis of Flag-tagged B7H3 ubiquitination with the indicated antibodies. d The indicated cell lines were treated with MG132 for 6 h. Flag-tagged B7H3 WT or its NQ mutants were pulled down by anti-Flag beads in the indicated cell lines, followed by western blotting to detect HRD1 and SEL1L. e MDA-MB-231-B7H3KO-N91/104/309/322Q cells were transiently transfected with HRD1 siRNA or SEL1L siRNA for 48 h. Then the cells were treated with CHX at indicated intervals. The intensity of B7H3 protein was quantified using ImageJ software. f MDA-MB-231-B7H3KO-N91/104/309/322Q cells expressing sgRNAs targeting HRD1 or SEL1L were treated with CHX at indicated intervals. The intensity of B7H3 protein was quantified using ImageJ software. g MDA-MB-231-B7H3KO-N91/104/309/322Q cells expressing sgRNAs targeting HRD1 or SEL1L were treated with MG132 for 6 h. Immunoprecipitation analysis of Flag-tagged B7H3 ubiquitination with the indicated antibodies. The p-value in (e, f) was determined by a two-tailed unpaired Student’s t-test. Error bars represent mean ± SD. Data in (b–g) are representative of three independent experiments.
