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Fig. 4 Effect of ezetimibe on CAV1–ANX2 complex. (A) Effects of ezetimibe on complex formation in zebrafish embryos. (Top) Immunoblot with CAV IgG. (Bottom) Immunoblot with ANX2 IgG. Lanes are identical in both blots. Embryos (48 hpf) were lysed and subjected to IP followed by SDS/PAGE (20 embryos per lane), and immunoblotted. Lane 1, CHO cells (control); lane 2, untreated embryos, IP with CAV IgG; lane 3, untreated embryos, IP with ANX2 IgG; lane 4, embryos soaked in 100 μM ezetimibe from 24 hpf to 48 hpf and subjected to IP with CAV IgG; lane 5, embryos soaked in ezetimibe as in lane 4 (IP with ANX2 IgG). (B) Immunoblots of mouse enterocytes. (Upper) Immunoblot with CAV IgG. (Lower) Immunoblot with ANX2 IgG. Lanes are identical in both blots. Ezetimibe-treated mice were administered the drug orally (1 mg⋅kg-1⋅d-1) for 4 weeks immediately before being killed (n = 12 mice per group). Mice that were either untreated or ezetimibe-treated were fasted for 4 h and killed; enterocytes were then isolated from the jejunum by the low-temperature method (31). Enterocytes were lysed, and the cytosol was subjected to IP followed by SDS/PAGE and immunoblotted with the same IgG used for precipitation. Lane 1, C57BL/6 mouse; lane 2, C57BL/6 mouse treated with ezetimibe; lane 3, LDL-receptor (LDLr) mutant mouse; lane 4, LDL-receptor mutant mouse plus ezetimibe; lane 5, C57BL/6 mouse on a Western diet; lane 6, C57BL/6 mouse on a Western diet plus ezetimibe. (C) Serum cholesterol measurements for the mice in B. (D) IP of CAV1 or ANX2 coprecipitates cholesterol. Enterocytes from C57BL/6 mice fasted for 4 h were isolated as in B and treated with 10 μM ezetimibe for 2 h at 37°C. The cytosol was subjected to IP, and the amount of cholesterol was determined by gas chromatography. (E) IP of CAV1 but not ANX2 or cyclophilin A coprecipitates ezetimibe. CACO-2 cells were treated as in D and subjected to IP; the amount of ezetimibe was determined by gas chromatography-mass spectrometry.