p110-β positively regulates PtdIns(3)P level and Vps34 catalytic activity. (A) β^+/+ and β^−/− MEFs stably expressing GFP-FYVE were observed under a deconvolution fluorescence microscope. Representative images (left) and quantification of GFP-FYVE puncta per cell (right) are shown. Bar, 20 µm. ***, P < 0.0001; n > 20; error bars = SEM. (B) Total cellular lipids were extracted from β^+/+, β^−/−, and β^+/+ MEFs treated with 1 mM 3-MA for 12 h. The lipid extracts were subjected to protein–lipid overlay analysis for PtdIns(3)P content. PtdIns(3)P standard and other PtdIns species were used as controls. Data shown are representative of three independent experiments. Quantification of total cellular PtdIns(3)P normalized against total protein is shown on the right (n = 3 for β^+/+ and β^−/−; n = 2 for β^+/+ +3-MA; **, P < 0.005; error bars = SEM). (C) β^+/+ and β^−/− MEFs were stained with antibodies against PtdIns(3)P or PtdIns(3,4,5)P₃, and subjected to flow cytometry analysis. (D and E) HEK293T cells were transfected with indicated plasmids. 48 h after transfection, cell lysates were subjected to immunoprecipitation using Myc antibody-conjugated agarose (D) or GFP antibody and protein A–conjugated agarose (E). 75% of the precipitates were assayed for Vps34 catalytic activity, and 25% of the precipitates were immunoblotted with indicated antibodies. Vps34 activity is calculated as the amount of PtdIns(3)P generated normalized against the amount of Vps34 present in the precipitates. IP reaction using IgG isotype control showed no activity over the background scintillation readings. Representative autoradiographs of the in vitro Vps34 assay results and the corresponding immunoblots are shown on the left. Quantification of Vps34 activity and Beclin 1–associated Vps34 activity are shown on the right. Data shown are average of three independent experiments ± SEM. *, P < 0.05. (F) Purified p110-β/p85-α was assayed under p110-β or Vps34 assay conditions. Results shown are the average of duplicate experiments.