Figure 4.

Effect of compound 28 on the photoaffinity labeling and ATPase activity of P-gp. (A) Compound 28 inhibits the photoaffinity labeling of P-gp with [¹²⁵I]-IAAP. Crude membranes of High Five insect cells expressing P-gp (65 μg protein/100 μL) in 50 mM MES-Tris pH 6.8 were incubated with increasing concentrations of compound 28 (0–20 μM), at 37 °C for 10 min. Samples were then transferred to 4 °C bath, and 4–5 nM [¹²⁵I]-IAAP was added under subdued light. The samples were photo-cross-linked with [¹²⁵I]-IAAP as described previously.³⁶ A representative autoradiogram from one of two independent experiments is shown. In the graph, points represent the average of two independent experiments. The data were fitted (R² = 0.94) with a one-phase decay equation using GraphPad Prism 6.01. (B) Compound 28 stimulates the basal ATPase activity of ABCB1. Crude membranes of High Five insect cells expressing P-gp (10 μg protein/100 μL) were incubated with increasing concentrations of compound 28 (0–10 μM), in the presence and absence of sodium orthovanadate (0.3 mM), in ATPase assay buffer as described previously.³⁷ The data obtained with compound 28 up to 2.5 μM concentration are shown in (B). Points with error bars represent the mean and SEM of three independent experiments. The data were fitted (R² = 0.92) with the Michaelis–Menten equation using GraphPad Prism 6.01.