Enhanced chemi luminescence (ecl)

pII cells were cultured in 6-well plates to 80% confluence and then serum-starved overnight before addition of either vehicle or EGF (50 ng/ml). After 30-min exposure cells were harvested by scraping into 300 μl of lysis buffer containing 50 mM HEPES, 50 mM NaCl, 5 mM EDTA 1% Triton X-100, 100 μg/ml PMSF, 10 μg/ml aprotinin, and 10 μg/ml leupeptin. Protein was determined by the standard Bradford assay and 6 μg were mixed with an equal volume of 2X SDS and heated at 90°C for 10 min. Lysates were loaded onto a 10% SDS-polyacrylamide gel and electrophoresed at 150 V for 1 h. Proteins were transferred to a nitrocellulose membrane and blocked with 2% BSA for 1 h before being incubated overnight at 4°C with either total or pAkt antibody (1/600 dilution), pERK1/2 antibody (1/1,000 dilution), Na_v1.5 antibody (1/100 dilution), or actin antibody (1/1,000 dilution) prepared in 2% BSA. The membrane was washed and incubated with anti-HRP-conjugated secondary antibody (1/500 dilution) for 1 h, developed with Super Signal ECL and visualized with Kodak X-ray film.

Tissue lysates from adult P-Rex1^+/+ and P-Rex1^−/− mice were prepared as previously described (6 (link), 10 (link)). Proteins from these and other lysates, prepared as described below, were denatured in boiling Laemmli sample buffer, resolved by SDS-PAGE, and transferred onto PVDF by wet transfer. Membranes were blocked in TBS, 0.05% Tween 20 containing 5% nonfat milk or 1% (w/v) BSA, as appropriate. Primary antibodies were used as previously described: myc and EE mouse mABs (Babraham Monoclonal Antibody Unit) (1 (link), 10 (link), 26 (link)), P-Rex1 mouse mAB 6F12 (from Prof Marcus Thelen, Institute for Research in Biomedicine, Bellinzona, Switzerland) (6 (link)), rabbit pABs Norbin C1, C3, N4, and N7 (from Dr. Kei Maruyama, Tokyo Institute of Psychiatry, Japan) (33 (link)), GST and β-actin mouse mABs (Sigma), HRP-coupled myc-AB P/N 46-0709 (Invitrogen), and Rac1 mouse mAB (Millipore 05-389) (6 (link)). Secondary antibodies used were HRP-conjugated goat anti-mouse IgG (Bio-Rad) or goat anti-rabbit IgG (Santa Cruz). Detection was done with Amersham Biosciences ECL or ECL-Plus and Kodak x-ray film. Where required, membranes were stripped in 25 mm glycine (pH 2.0), 1% SDS and reprobed. For densitometric analysis, films were scanned and band intensities quantified using ImageJ. To test total protein loading, PVDF membranes were stained with Coomassie after blotting.

Cells were lysed in RIPA buffer (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% SDS, 1% sodium deoxycholate, and 1 mM EDTA) containing complete EDTA-free protease inhibitor cocktail (Roche), 1 mM PMSF, and phosphatase inhibitors (5 mM sodium orthovanadate). Protein lysates were separated by SDS/PAGE, electrically transferred to a Hybond ECL nitrocellulose membrane (Amersham Biosciences, Freiburg, Germany), immunoblotted with antibodies and visualized by ECL (Kodak, Rochester, NY, U.S.A.). Protein levels were normalized to the level of β-actin. The following primary antibodies were used for immunoblotting: anti-Actin (Abcam, Cambridge, MA, U.S.A.), anti-MAPK, anti-IKK-β, anti-PARP, anti-caspase9 (Cell Signaling Technology, MA, U.S.A.), anti-CCND1, anti-CDK4 (Santa Cruz Biotechnology, CA, U.S.A.).

Whole-cell lysates were prepared in RIPA buffer, the composition of which was described in J Lian et al.,^{8 (link)} separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto Hybond enhanced chemiluminescence (ECL) Nitrocellulose membrane (Amersham Biosciences, Freiburg, Germany), immunoblotted with antibodies and visualised by ECL (Kodak, Rochester, NY, USA). Protein levels were normalised to GAPDH. The information regarding the antibodies is provided in Supplementary Table S4.