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Fig. 3 Effect of reducing ANX2b protein in zebrafish larvae. Newly fertilized embryos (1–8 cell stage) were injected with anx2b MO and allowed to develop. (A) Larvae (5 d postfertilization) were fed NBD-cholesterol as described (2) then photographed. Uninjected larvae concentrate NBD-cholesterol in the gall bladder (arrowhead) and intestine (arrow). (B) IP and immunoblot to determine the persistence of anx2b MO effect. Embryos were injected with anx2b MO, collected, lysed, subjected to IP, and immunoblotted as described in the legend to Fig. 2. Uninjected control embryos are 48 hpf. For the 120-h sample, embryos were fed NBD-cholesterol and sorted into low- and high-intestinal fluorescence groups before lysis and IP. Data are representative of three to five experiments with 20–30 larvae per group. (Scale bar, 500 μm.) (C) Effect of anx2b MO on lipid composition. Embryos were injected with anx2b MO, allowed to develop for 72 h, then collected. The total lipid was then collected, and the amount of cholesterol, cholesteryl ester, and triglycerides was determined for injected (open bars) and control uninjected (closed bars) embryos. Each bar represents the mean of six measurements, 20 embryos per measurement. Differences between injected and control embryos for both cholesterol and cholesteryl ester are statistically significant (P < 0.05). (D) Rescue of cholesterol by anx2b mRNA. Uninjected, anx2b MO-injected, and anx2b MO/anx2b mRNA coinjected embryos were collected at 48 hpf, and the cholesterol per embryo was determined by mass spectrometry. Each point is the average of three separate batches of 20 embryos each. The MO-injected value shows a statistically significant difference from both the uninjected and MO plus RNA values (Tukey honestly significant difference test; P < 0.01), whereas uninjected and MO plus RNA values do not show statistically significant difference from each other. (E) Immunoblot of embryos from the same experiment as D to demonstrate that rescue of cholesterol concentration by anx2b mRNA is coincident with rescue of CAV1–ANX2b protein complex.