Fig. 5
From: Coordinate regulation of mutant NPC1 degradation by selective ER autophagy and MARCH6-dependent ERAD
I1061T is degraded by ER-phagy. a CTRL, I1061T, or P1007A/T1036M NPC1 fibroblasts were treated with the following siRNAs: non-targeting (NT), beclin-1 (Bec1), p97, and FAM134B (FAM) at t = 0 and t = 24 h. Lysates were collected at t = 48 h and NPC1 levels analyzed and quantified below. b I1061T NPC1 fibroblasts were treated for 2 sequential days with non-targeting siRNA (siNT) or siRNA FAM134B (siFAM134B), then treated with cycloheximide and serum starved. NPC1 protein levels were analyzed by western blot, quantified at right. c Lysates from a were digested with endoglycosidase H (E), PNGaseF (P), or not treated (NT) and then analyzed by western blot. d I1061T NPC1 fibroblasts were treated with siRNAs as in a. At t = 48 h, cells were stained with filipin. Intensity quantified at right. Scale bar = 50 μm. Data are mean ± s.e.m. from a 3–6; b, c 3 independent experiments. n.s., not significant, *P ≤ 0.05. **P ≤ 0.01 by a, b ANOVA with a Tukey or b Bonferroni posthoc test; c Student’s t-test. *P ≤ 0.05, a (I10) F = 10.37; b F = 7.45
