a, Top: Western blot analysis of the Tmem97:NPC1 interaction in HEK293T cells overexpressing Tmem97-Myc-FLAG. Cells were treated with indicated concentrations of 20(S)-OHC for 24 h, then endogenous NPC1 was captured from cell lysates using overexpressed Tmem97 as bait. Bottom: Quantitative Western blot analysis of NPC1 and Tmem97 in the sample input and after co-immunoprecipitation. Data are the average of 3 biological replicates ± s.e.m. Statistical significance, evaluated by two-way ANOVA with Dunnett’s multiple comparisons test, is shown, **Padj = 0.0056; n.s. = not significant.
b, Top: Western blot analysis of the Tmem97:NPC1 interaction in HEK293T cells overexpressing Tmem97-Myc-FLAG. Cells were treated with DMSO or 10 μM 20(S)-OHC for 24 h, then overexpressed Tmem97 was captured from cell lysates using endogenous NPC1 as bait. Bottom: Quantitative Western blot analysis of overexpressed Tmem97 and NPC1 in the sample input and after co-immunoprecipitation. Data are the average of 4 biological replicates ± s.e.m. Statistical significance, evaluated by a two-tailed unpaired t-test between indicated conditions, is shown, *p = 0.0217; n.s. = not significant.