Figure S2.
Active recombinant PTEN does not dephosphorylate phospho-Akt S473 or phospho-FAK Y397. (a) AML12 mouse hepatocytes were treated with 10 nM insulin for 15 min and immunoprecipitated with anti-Akt antibody. The immunoprecipitates were incubated either with or without 0.1 µg/µl active recombinant PTEN (SignalChem; catalog number P23-20G-10) for 30 min in the reaction buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 50 µM MgCl2, 0.05% Tween-20, and 0.2% 2-mercaptolethanol. After incubation, the samples were analyzed by immunoblotting for phospho-Akt S473 (pAkt S473) and Akt. (b) Human fibroblasts were cultured on gelatin-coated dishes overnight and immunoprecipitated with anti-focal adhesion kinase (FAK) antibody. The immunoprecipitates were treated either with or without active recombinant PTEN as described above. The treated samples were then analyzed by immunoblotting for phospho-FAK Y397 (pFAK Y397) and FAK. IP, immunoprecipitation.