Anti rhoa

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.

Protein lysates were separated by SDS-PAGE, and electrophoretically transferred to PVDF (polyvinylidene difluoride) membrane. Then, where relevant, the blots were probed with the primary antibodies as labeled in the corresponding Figs, followed by HRP (horseradish peroxidase)-labeled goat anti-mouse or goat anti-rabbit IgG, and the signals were detected using enhanced chemiluminescence (ECL). β-actin was used as a protein loading control. For origin and description of all antibodies used in this study see Table S3 in File S1.
Rho-GTPase activation assay was carried out according to the methods described previously [34] (link). In brief, PAK1 PBD-agarose (for isolating Rac1-GTP and cdc42-GTP) and rhotekinagarose (for isolating Rho-GTP) (Millipore) were used to pull down the GTP-bound form of Rho-GTPase according to the manufacturer’s manual. The levels of active Rac1, cdc42, and RhoA were detected by Western blot using specific polyclonal anti-Rac1 (1∶1000), anti-cdc42 (1∶250), and anti-RhoA (1∶375) antibody (Millipore).

Antibodies were obtained from: Cell Signaling [Danvers, MA, USA: anti‐phospho‐Src (tyr416), anti‐Src, anti‐phospho‐MLC (ser19), anti‐MLC, anti‐MYPT1, anti‐p115RhoGEF, anti‐RhoA, anti‐cofilin, anti‐phospho‐cofilin (ser3)], Millipore [anti‐phospho‐MYPT1 (thr696)], Sigma (anti‐rabbit IgG, anti‐mouse IgG) and Santa Cruz Biotechnology (Santa Cruz, CA, USA: anti‐ROCK2). All antibodies were used at 1:1000 dilution except for anti‐phospho‐MYPT1, anti‐rabbit IgG and anti‐mouse IgG, which were used at 1:5000. PP2 [4‐amino‐5‐(4‐chlorophenyl)‐7‐(dimethylethyl) pyrazolo[3,4‐d]pyrimidine] was obtained from Calbiochem; BK, Y16 (inhibitor of RGS‐domain containing RhoGEFs) and Y27632 (Rho‐kinase inhibitor) were obtained from Sigma. Rhotekin reagent and GTP‐γ‐S were obtained from Millipore. TGF‐β was obtained from R&D Systems (Minneapolis, MN, USA). Cell culture and western blot materials were from Cell Signaling, Invitrogen, GE Healthcare or Thermo Scientific. General reagents were from Sigma, Calbiochem or VWR (Radnor, PA, USA).