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. 2022 Dec 8;7(23):e160308. doi: 10.1172/jci.insight.160308

Figure 3. NPC1 protein sequence drives differential trafficking.

Figure 3

(A and C) NPC1-deficient mouse fibroblasts were electroporated with (A) human WT, human I1061T, and mouse I1061T and (C) human R934L and mouse R934L, and total NPC1 was analyzed by Western blot and quantified. (B) Mouse fibroblasts and human Hap1 cells deficient in NPC1 were electroporated with GFP, human WT-NPC1, human I1061T-NPC1, or mouse I1061T-NPC1 plasmids. Lysates were incubated with no treatment (NT) or digested with EndoH (E) or PNGase F (P) and then subjected to Western blot and quantified. (D) NPC1-deficient mouse fibroblasts were electroporated with human WT-NPC1, human R934L-NPC1, or mouse R934L-NPC1 plasmids, and lysates were treated as in B and quantified. (E) Hu-I1061T, Mu-I1061T, or Mu-I1061T containing the human glycosylation sites (Ms-I1061T+HuGlycans) were overexpressed and digested with E or P and quantified. Data are shown as the mean ± SEM from indicated number of independent experiments. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001 by (A, B, D, and E) ANOVA with Tukey’s post hoc test or (C) t test. (A and C) n = 3, F = 6.7, t = 3.8, df = 2,4. (B) Hap1, Mef; n = 4,4; F = 37.8, 104.5; df = 2. (D) n = 4; F = 95.5; df = 2, (E) n = 5, F = 248.2, df = 2. See complete unedited blots in the supplemental material.