PIP5Kγ silencing reduces AP-2 membrane–bound levels. (A) YTS cells were treated with PMA/ionomycin for the indicated times. Cell lysates were precipitated with normal mouse IgG or α-adaptin1/2 (AP-2) mAb and AP-2/Munc13-4 complexes formation was evaluated by immunoblot analysis as indicated (top panel). The same membrane was reprobed with anti–α-adaptin mAb as a oading control (bottom panel). One representative experiment of 3 performed is shown. (B) AP-2 immunoprecipitates were obtained from membrane and cytosolic fractions isolated from shRNA-ctr, shRNA-PIP5Kα, and shRNA-PIP5Kγ YTS cell populations and analyzed by immunoblot (top panel). Total lysates of the same samples were analyzed by immunoblotting with the indicated mAbs as purity and loading controls (middle and bottom panels). One representative experiment of 3 performed is shown. (C) Quantitative analysis of the ratio of AP-2 levels in the membrane and cytosolic fractions shows a significant difference (*P < .05) between shRNA-ctr and shRNA-PIP5Kγ populations. Means from 3 independent experiments ± SD are graphed.