(A and B) IP of HRD1 in HRD1P398L KI HEK293T cells to test its interaction with components of the ERAD complex, with quantitation shown in B. n = 3 per group. (C and D) Denaturing IP of HA-agarose in KI HEK293T cells expressing indicated variants transfected with a model substrate proAVP (G57S)-HA to measure substrate ubiquitination, with quantitation shown in D. n = 3 per group. (E and F) Denaturing IP of FLAG-agarose in HRD1–/– HEK293T cells transfected with indicated HRD1 variants, with or without 10 μM MG132 for 2 hours, to measure HRD1 ubiquitination, with quantitation shown in F. n = 3 per group. n, individual cell samples. Data are represented as means ± SEM. **P < 0.01; ***P < 0.001; ****P < 0.0001, 2-tailed Student’s t test (B); 1-way ANOVA followed by Dunnett’s multiple-comparisons test (D and F).