Phosphoinositide 3-kinase activity assay

PI3K activity was determined as described previously [7]. Briefly, RMCs cultured in 6-well dishes were serum-deprived for 24 h, 2 x10⁵ cells received different treatments and were washed twice with ice cold phosphate-buffered saline and lysed with 1 mM lysis buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 1% Nonidet P-40, 10% glycerol, 1mg/ml bovine serum albumin, 20 mM Tris, pH 8.0, 2 mM orthovanadate). Cell extracts were incubated with 2 μg of anti-p85 antibody overnight at 4°C. The immunocomplex was precipitated with 50 μl of protein A-Sepharose for 1 h at 4°C and washed three times with lysis buffer, twice with LiCl buffer (0.5 M LiCl, 100 mM Tris, pH 7.6), and twice with TNE buffer (10 mM Tris, pH 7.6, 100 mM NaCl, 1 mM EDTA). The immunocomplex was preincubated with 10 μl of 20 mM Hepes (pH 7.4), containing 2 mg/ml propidium iodide (Sigma) on ice for 10 min. Kinase reaction was performed by adding 40 μl of reaction buffer (10 μCi of [γ-³²P]ATP, 20 mM Hepes, pH 7.4, 20 μM ATP, 5 mM MgCl₂) at room temperature for 15 min. Added 100 μl of 1 M HCl extracted with 200 μl of a 1:1 mixture of chloroform and methanol to stop the reaction. The radiolabeled lipids were separated by thin-layer chromatography and visualized by phosphorimaging.