Fig. 9: US9 SP arrests MICA*008 maturation, indirectly leading to ERQC-mediated degradation.

a Schematic representation of the effect of kifunensine (KIF) treatment (red line) on the ER quality control (ERQC) pathway. KIF inhibits mannosidases, preventing glycosylation trimming of misfolded glycoproteins and subsequent ER-associated degradation (ERAD). Therefore, KIF treatment should cause the accumulation of untrimmed misfolded glycoproteins. b RKO MICA*008-HA cells expressing EV or US9 were untreated (−) or treated (+) with 200 µM KIF and lysed. Lysates were mock-treated (Mock), or digested with endoH or with PNGaseF, and blotted using anti-MICA, with anti-vinculin as loading control. Annotated are the glycosylated forms of MICA*008 (Gly⁺), and deglycosylated MICA*008 forms with or without the GPI anchor (GPI⁺-Gly⁻ and GPI⁻-Gly⁻, respectively). * Endogenous MICA*007:01 (42 kDa). Red squares highlight PNGaseF-digested forms used for protein quantitation. c Deglycosylated MICA*008 levels were normalized to the loading control and quantified relative to the mock EV control. The fold change in levels following KIF treatment was calculated. Data show mean ± SEM for four independent experiments. A two-tailed unequal variance Student’s t-test was used to compare the fold change in EV vs. US9-expressing cells (p = 0.011). *p < 0.05. d The indicated cells were untreated (−) or treated (+) with KIF as described. Lysates were obtained, digested with PNGaseF, and blotted using anti-MICA, with anti-vinculin as loading control. Annotated are GPI-anchored (GPI⁺-Gly⁻) and unanchored (GPI⁻-Gly⁻) deglycosylated MICA*008 forms. e MICA*008 levels shown in (d) were normalized to the loading control and quantified relative to the untreated EV control. The fold change in total MICA*008 protein levels following KIF treatment was calculated. Data show mean ± SEM for four independent experiments. A one-way ANOVA was performed with a significant effect at the p < 0.05 level for all conditions [F(4,15) = 15.36, p = 3.4 × 10⁻⁵]. A post hoc Dunnett’s test was used to compare the fold change for EV (dashed line) to that of US9 and the mutants. Red columns show significant KIF-induced increase in total MICA*008 levels. **p < 0.01, NS non-significant. See also the Source data file for experimental data and full statistics.