Fig. 2
From: Coordinate regulation of mutant NPC1 degradation by selective ER autophagy and MARCH6-dependent ERAD
I1061T NPC1 is degraded in part by MARCH6-dependent ERAD. a Tamoxifen-inducible Sel1L null mouse embryonic fibroblasts expressing WT NPC1 were treated with vehicle (NT) or 200 nM tamoxifen (Tam) for 48 h. Cells were also treated with vehicle (Veh) or 10 µM MG132 (MG) during the last 24 h to determine if Sel-1L deficiency alters the pool of WT NPC1 trafficking to the proteasome. NPC1 protein level quantified below. b Over-expression of FLAG, GP78-FLAG, Der1-HA, and HRD1-FLAG in I1061T NPC1 fibroblasts. Cells were transfected at t = 0 and t = 24 h and lysates collected at 48 h. Transgene expression confirmed using antibodies against FLAG and HA. NPC1 quantified at right, normalized to I1061T NPC1 fibroblasts transfected with FLAG. c I1061T NPC1 fibroblasts were treated with the following siRNAs: non-targeting (NT), GP78, Der1, and HRD1 at t = 0 and t = 24 h. Lysates were collected at 48 h. NPC1 quantified at right, normalized to I1061T NPC1 fibroblasts transfected with NT siRNA. d, e CTRL and I1061T NPC1 fibroblasts were treated with d non-targeting or MARCH6 siRNA (shown are two independent experiments for I1061T), e vehicle (Veh) or 10 μM eeyarestatin I (Eey). Lysates were blotted for NPC1 (quantified at right). Data are mean ± s.e.m. a–e N = 3–4, b, c N = 5–6, d N = 4–6. a–c One-way ANOVA with Tukey’s posthoc test, a F = 8.147. d, e Student’s t-test, d (I10) t = 4.8, e t = (CTRL) 2.69, (I10) 3.16. n.s. = not significant, *P ≤ 0.05, ***P ≤ 0.001
