Fig. 1. centaurin-₂ interacts with PI 3,4-P₂ and PIP₃ via the C-terminal PH domain in vitro. COS7 cells were transiently transfected with GFP, GFP-centaurin-₂ or its mutants [the N-PH mutant (R152C), the C-PH mutant (R276C) and the double mutant (R152C/R276C)]. After 2 days, the cells were lysed and the lysates were incubated with the indicated biotinylated PI lipids coupled to avidin beads either in the presence (B) or absence (A,C) of water soluble non-biotinylated PI lipids for 2 hours at 4°C. Protein that remained bound to the PI beads after washing with 0.5% NP-40 was analysed by immunoblotting with an anti-GFP antibody.