Fig. 4: NPC1 increases protein stability of TGFBR1 and inhibits its ubiquitination.

a, b qPCR analysis of NPC1 and TGFBR1 mRNA levels in NPC1-overexpression PLC/PRF/5 (a) or NPC1-knockdown HepG2 (b) cells. c, d PLC/PRF/5 cells with/without stable overexpression of NPC1 (c) or HepG2 cells with/without stable knockdown of NPC1 (d) were treated with CHX for indicated times and then analyzed by western blot. e HepG2 cells with or without stable knockdown of NPC1 were treated with vehicle, MG132 (10 μM), or NH₄Cl (10 mM) for 12 hours. Cell lysates were subjected to immunoblot with TGFBR1 or NPC1 antibody. f, g TGFBR1-mCherry-His stable overexpression PLC/PRF/5 (f) and HepG2 (g) cells with or without NPC1 overexpression (f) or knockdown (g) were pretreated with MG132 (10 μM) for 8 hours before collection. Then TGFBR1-mCherry-His was pulled down by Ni-NTA and immunoblotted with anti-K48-Ubiquitin, anti-K63-Ubiquitin and anti-Ubiquitin antibody. Data are presented as the mean ± s.e.m. n = 3 (a–e) biologically independent samples. Statistical significance was determined by two-tailed unpaired Student’s t-test (a, b, e) or two-way analysis of variance (ANOVA) (c, d). All experimental data were verified in three independent experiments. Source data are provided as a Source Data file.