Fig 1: SEL1LC141Y variant causes proteasome-mediated self-destruction of SEL1L-HRD1 ERAD complex.(A) Western blot analysis of SEL1L and HRD1 in WT and SEL1LC141Y patient fibroblasts treated with and without 10 μM MG132 for 2 hours, with quantitation shown in Supplemental Figure 9A. n = 6–8 (WT); n = 3–4 (C141Y). (B and C) Western blot analysis of ERAD proteins and endogenous ERAD substrates in WT or KI HEK293T cells with and without HRD1–/– or RNF5–/–, with quantitation shown in C and Supplemental Figure 9, C and E (n = 3–9 per group). (D) Western blot analysis of HRD1 in WT or KI HEK293T cells with and without HRD1–/– using 2 different SEL1L antibodies, with quantitation shown in Supplemental Figure 9B (n = 3–6 per group). (E and F) Cycloheximide chase analysis of ERAD proteins in WT and KI HEK293T cells with and without HRD1–/–, with quantitation shown (F) (n = 3–6 per group). OS9 1 indicates isoform OS-9.1; OS9 2 indicates isoform OS-9.2. Both bands were quantitated together. (G) Reducing and nonreducing SDS-PAGE and Western blot analysis of HMW aggregates of SEL1L in KI HEK293T cells with and without HRD1–/– (representative of 2 repeats). Data are represented as means ± SEM. **P < 0.01; ***P < 0.001; ****P < 0.0001, 1-way ANOVA followed by Tukey’s post hoc test (C); 2-way ANOVA followed by Tukey’s multiple-comparisons test (F).