Fig. 5: The regulatory subunit p85 of class I_A PI3K is a substrate of AEP.

A p110ɑ coimmunoprecipitated with AEP. Total cell lysates of MDA-MB-231 cells were used for anti-AEP immunoprecipitation and immunoprecipitants were analyzed by immunoblotting with antibodies specific to p110ɑ and AEP. Biological independent replicates. *, non-specific bands. B Cleavage of p85 correlated with AEP activation. MDA-MB-231 cells were treated with 5 µM Chloroquine (CQ) for indicated times. AEP activation and p85 cleavage were analyzed by immunoblotting with antibodies specific to AEP, p85, and p62. C, D Subcellular fractionation showing membrane localization of p85 truncate and AEP. ME and CE were analyzed by immunoblotting with indicated antibodies. Rab7, LAMP2, LC3, and cathepsin D are loading controls. The immunoblots shown are from representative experiments. E MDA-MB-231 cells were immunostained with antibodies specific to p85 and AEP, and examined by confocal microscopy. Arrow denotes p85 and AEP colocalization. F AEP localized on the outer membrane of and inside lysosomes. 3D images of cells stained with LAMP02 and AEP were re-constructed from confocal microscopy serial optical Z-sections. AEP cleaved p85 in vitro. GST-p85 was incubated with increasing amount of AEP in vitro cleavage experiment. Cleavage of p85 was visualized by Coomassie blue staining (G) or by immunoblotting with antibody specific to p85 (H) after SDS-PAGE separation. The results shown are from representative experiments.