ABCB1 ATPase Assay

The ABCB1 ATPase activity was measured based on vanadate-sensitive principle as previously described (Zhang et al., 2016). The High Five insect cells membrane vesicles (BD Biosciences, San Jose, CA, USA) were first incubated for 5 min in ATPase assay buffer at 37°C with or without 0.3 mM vanadate. Then peptide HX-12C at a concentration of 0 to 40 μM was adding to the assay buffer and incubated at 37°C for 3 min. After adding 5 mM Mg-ATP with a total volume of 0.1 mL, the ATPase reaction was initiated until stopped by adding 100 μL 5% SDS solution after incubation at 37°C for 20 min. The amount of inorganic phosphate (IP) was used to calculate the ATPase activity of ABCB1, detected at 800 nm using a spectrophotometer.