Identification of FABP7 key residues involved in cholesterol transport and binding. (A–C) FABP7 KO2 cells were transfected with the indicated plasmids expressing various FABP7 mutants tagged with Flag for 48 h and harvested. (A) Immunoblotting analysis showing the expression of indicated FABP7 mutants. (B) Cells were fixed and stained with filipin (magenta) and the antibody against Flag (green). Scale bars, 10 µm. (C) Quantification of relative fluorescence intensity of filipin in B. The filipin fluorescence intensity of FABP7 KO2 cells transfected with the plasmid expressing wild-type (WT) FABP7 is defined as 1 and used as the reference for comparison. Data are presented as median with interquartile range (n = 583 cells from three independent experiments). Mann–Whitney U test. (D) The cholesterol binding activities of FABP7 protein. Data are presented as mean ± SEM (from three independent experiments). (E) Binding of ³H-cholesterol to WT FABP7 recombinant protein and the indicated mutants. Equal amounts of proteins were used in the reaction. CPM measured for ³H-cholesterol bound to WT FABP7 protein is defined as 1 and used as the reference for comparison. Data are presented as mean ± SEM (from three independent experiments). Student’s unpaired two-tailed t test with Welch’s correction. (F) Binding of ³H-cholesterol to FABP7 in the presence of different fatty acids. CPM measured for ³H-cholesterol bound to FABP7 in the absence of (−) the indicated competitors is defined as 1 and used as the reference for comparison. Data are presented as mean ± SEM (from three independent experiments). Student’s unpaired two-tailed t test with Welch’s correction. (G) Binding of ³H-cholesterol to FABP7 in the presence of increasing concentrations of oleic acid or cholesterol. CPM measured for ³H-cholesterol bound to FABP7 in the absence of (−) the indicated competitors is defined as 1 and used as the reference for comparison. Data are presented as mean ± SEM (from three independent experiments). Student’s unpaired two-tailed t test with Welch’s correction. (H) Binding of ³H-oleic acid to WT FABP7 and the indicated mutants. Equal amounts of proteins were used in the reaction. CPM measured for ³H-oleic acid bound to WT FABP7 is defined as 1 and used as the reference for comparison. Data are presented as mean ± SEM (from three independent experiments). Student’s unpaired two-tailed t test with Welch’s correction. Source data are available for this figure: SourceData F5.