2.8. ATPase assay

The vanadate‐sensitive ATPase activity of ABCB1 was quantified with similar protocol described previously [³⁸]. In brief, we used ABCB1‐membrane to hydrolyze ATP and measure the inorganic phosphate (P_i) production. Reactions took place in assay buffer containing different types of ATPase inhibitors (1 mmol/L ouabain, 5 mmol/L sodium azide, 0.01 mol/L MgCl₂, 2 mmol/L EGTA, 0.05 mol/L MES [pH 6.8], 0.05 mol/L KCl, and 2 mmol/L DTT). Then sitravatinib was incubated with membrane vesicles for 3 min. The ATP hydrolysis was initialized by adding 5 mmol/L of Mg‐ATP. After incubation at 37°C for 20 min, 5% SDS solution was added to terminate the reaction. P_i was measured at 880 nm in a spectrophotometer (Bio‐Rad, Hercules, CA, USA).