Fig. 7: NPC1L1 associates with membrane in a cholesterol-dependent manner.
a NPC1L1-CT67 recombinant protein was purified and co-sedimented with serial liposomes with different concentrations of cholesterol (n = 2). After incubation and centrifugation, supernatant (S) and pellet (P) were collected and subjected to immunoblotting analysis. b The purified NPC1L1-CT67 recombinant proteins (WT, ΔA1272LAL and A1272LAL → 4E mutant) were analyzed using liposome co-sedimentation assay (n = 2). c Interaction of LIMA1-FLAG and indicated NPC1L1 variants (WT, ΔA1272LAL and A1272LAL → 4E) was detected in the presence or absence of BA mixture (n = 2). d Representative confocal images of surface NPC1L1 of CRL1601 cells overexpressing NPC1L1-3×Myc-EGFP or A1272LAL deletion (ΔA1272LAL) or mutation (A1272LAL → 4E) variants following BA mixture treatment over time. Scale bar, 10 μm. e The relative intensity of surface NPC1L1 in (d) was analyzed. The average intensity of cells overexpressing WT NPC1L1-3×Myc-EGFP with 120 min-treatment of BA mixture was defined as 1. Values were presented as mean ± SD (n = 3 independent trials, 100 cells/trial). One-way ANOVA with Tukey post hoc test, ***P < 0.001. f A working model illustrating the role of BAs involved in NPC1L1-mediated cholesterol transport. CE cholesteryl ester, ER endoplasmic reticulum, ERC endocytic recycling compartment. Source data are provided as a Source Data file.
