Malachite green phosphatase assay

INPP4B phosphatase assays were performed using the Malachite Green Phosphatase Assay (Echelon, Cat # K-1500) according to the manufacturer’s instructions. Recombinant purified INPP4B protein (Andrew Ellisdon) was preincubated with recombinant purified Rab7 protein (Abcam, Cat # 103507) for 30 min gently shaking at 37 °C in phosphatase buffer. Samples were placed on ice for 2 min. About 50 µM PI(3,4)P₂ diC8 (Echelon, Cat # P-3408) was added and reactions were incubated at 37 °C for 20 min. Samples were placed on ice, and 20 µL of each sample was loaded into a 96-well plate. A total of 80 µL of malachite green reagent was added and after 15 min, the color development was measured at 655 nm using a PheraStar plate reader (BMG Labtech). Phosphate standards were used to generate a four-parameter non-linear regression standard curve to calculate phosphate release.