Skip to main content
. 2005 Apr;25(7):2593–2606. doi: 10.1128/MCB.25.7.2593-2606.2005

FIG. 3.

FIG. 3.

Loss of all p85α and p85β gene products is associated with increased expression of p55γ but reduced overall PI3K activity. (A) p85 protein levels. HA-p55γ was transfected into a mutant cell pool (M1). The next day, growing MEFs of the indicated genotypes were lysed and equal amounts of protein were subjected to SDS-PAGE and immunoblotted with anti-p85pan (upper panel) or anti-p55γ antisera (lower panel). Panel A shows one out of three independent experiments with similar results. (B) p85-associated PI3K activity. Exponentially growing MEFs of the indicated genotypes were lysed, and equal amounts of protein were immunoprecipitated with anti-p85pan antibody. The immunoprecipitates were subjected to in vitro PI 3 -kinase assay with phosphoinositol (PI) as a substrate. The left panel in panel B shows a representative radiograph with one mutant cell pool (M2) and one control cell pool (C). A duplicate set was pretreated with 100 nM wortmannin (WM) for 20 min to inhibit the reaction. The right panel in panel B shows a quantification of three independent experiments described in the left panel, using three different mutant cell pools (M) and one control cell pool (C). Results are means ± standard error of the relative PI3K activity. (C) p110α protein levels. Exponentially growing MEFs of the indicated genotypes were lysed, and equal amounts of protein were immunoprecipitated with an antibody specific for p110α. The immunoprecipitates were subjected to SDS-PAGE and probed with anti-p110α antibody. Equal loading was verified by detecting similar amounts of Erk on the same membrane(data not shown). Panel 3C shows a representative immunoblot with one control pool (C) and two mutant pools (M1 and M3). (D) p110α-associated PI3K activity. Exponentially growing MEFs of the indicated genotypes were lysed, and equal amounts of protein were immunoprecipitated with anti-p110α antibody. The immunoprecipitates were subjected to in vitro PI3K assay with PI as a substrate. The left panel in panel D shows a representative radiograph on one control pool (C) and one mutant pool (M2). The right panel in panel D shows a quantification of three independent experiments described in the left panel, using three mutant pools (M) and one control pool (C). Results are means ± standard error of the relative PI3K activity.