Anti flag m2 affinity gel

The cells were collected and washed in cold 1 × PBS and lysed using RIPA (Millipore) with 1 mM PMSF, and 1 × protease inhibitor cocktail (Calbiochem) as per manufacturer’s protocol after transfection. The whole-cell lysates were incubated for 4 h at 4 °C with anti-Flag M2 Affinity Gel or GFPTrap (ChromoTek) in ratio recommended by manufacturer for immunoprecipitation.

Cells were harvested, lysed, and briefly sonicated. After centrifugation at 12,000 × g for 10 min at 4 °C, the supernatants (whole-cell lysates) were collected. For immunoprecipitation of Flag-tagged proteins or GFP-tagged proteins, cells were harvested and lysed after transfection and whole-cell lysates were incubated with anti-Flag M2 Affinity Gel or GFPTrap (ChromoTek) at 4 °C overnight. For immunoprecipitation of endogenous proteins, supernatants were sequentially incubated with PTEN antibodies overnight and protein A/G-agarose beads (Santa Cruz, CA) at 4 °C for 4 h. The precipitates were washed three times with immunoprecipitation buffer (50 mM Tris-HCl, PH 7.6, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM PMSF, and 1x protease inhibitor cocktail (Calbiochem)), boiled in sample buffer and subjected to Western blot analysis.