Alliance mini wl2m system

Whole cell extracts were prepared with NP-40 lysis buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% NP-40) containing protease inhibitors (SigmaFast, Sigma-Aldrich). Western blot analysis were carried out by standard methods with the exception of experiments reported in Fig. 5E and Supplemental Fig. S3 where chemioluminescence was detected with Alliance Mini WL2M system (Uvitec, Cambridge, UK). Membrane and cytosol enriched extracts were prepared with FractioPrep Cell Fractionation kit (BioVision, Mountain View, CA). Densitometric analysis of gel bands was carried out with ImageJ software (NIH, Bethesda, MD): the sum of band intensities in the cytoplasm and nuclear/membrane compartments was set as 100%. Anti-phospho-AKT (Ser473) (#4058), anti-AKT1 (#2938), anti-p42/44 (#9107), were purchased from Cell Signaling Technology (Danver, MA); anti-phospho-p27 (Thr157) (AF1555) and anti-phospho-p27 (Thr197/8) (AF3994) were from R&D Systems (Minneapolis, MN); anti-p27 (sc-528), anti-MYC (sc-40), anti-tubulin (sc-8035), anti-phospho-cofilin (sc-12912), anti-cofilin (sc-33779) were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-RhoA was from Millipore (Billerica, MA); anti-β-actin (clone AC-74, #A2228) was from Sigma-Aldrich.

Whole cell protein extracts were prepared by homogenizing cells in NP-40 lysis buffer (10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% NP-40) containing protease and phosphatase inhibitors. Western blot analysis was carried out by standard methods; chemioluminescence was detected with Alliance Mini WL2M system (Uvitec, Cambridge, UK). Anti-phospho-AKT (Ser473) (Rabbit, #4058, 1:1000) and anti-AKT1 (Rabbit, #2938, 1:1000) were purchased from Cell Signaling Technology (Denver, MA, USA); anti-actin (goat, sc-1616, 1:3000) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).