In vitro tyrosine kinase assay

In vitro tyrosine phosphorylation of SNX9 was determined by incubating 50 ng recombinant His–Src or His–FAK with 500 ng bacterially purified His–SH3PX1 in kinase reaction buffer (50 mM Tris HCl pH 7.4, 2 mM MgCl₂, 10 mM MnCl₂, 1 μM ATP, 10 μCi [32P]-ATP). Reactions were carried out at room temperature for various lengths of time (5–120 min), and then quenched with the addition of 5× SDS loading buffer.