Tris acetate mini gels

Cells were lysed in lysis buffer (150 mM NaCl, 30 mM TRIS, 0.1% Triton X-100, 10% glycerol and 1X protease inhibitor cocktail). Protein concentration was quantified using the BCA method (Pierce). 50 μg of total protein was resolved either on 4–12% NuPAGE^® Bis-Tris or 3–8% Tris-Acetate mini-gels (Invitrogen) and transferred to PVDF membrane. Membranes were blocked for 1 h at room temperature (RT) either with 5% non-fat dry milk or 5% Bovine serum albumin (BSA) in Tris-buffered saline containing 0.01% Tween 20 (TBS-T) and incubated overnight at 4 °C with primary antibodies. Chemiluminescence was detected using Novex® ECL substrate reagent and High performance film (GE Healthcare). β-actin, α-tubulin or GAPDH were used as loading controls. Antibodies for PDGFR-α (#5241), PDGFR-β (-#3169), GRB2 (#3972), pERKs (#9101), RAS (#8955), pAKT (#4060), totAKT (#2966), AKT2(#5239), pp53 (Ser15) (#9284), pRelA (#3033), totRelA (#6956), IKKβ (#8943), GAPDH (#3683) were purchased from Cell Signaling Technology. SOS1 (ab140621), IQGAP1 (ab86064), ERK2 (ab124362), p21 (ab109520), CDK1 (ab19), p53 (ab179477) antibodies were obtained from Abcam. MDM2 antibody was purchased from Calbiochem (OP115). RALA (sc-374462), Raf-1(sc-227) and β-actin (SC-47778) antibodies were purchased from Santa Cruz Biotechnology.

HLECs were lysed in ice-cold RIPA buffer (50 mM Tris, 150 mM NaCl, 1% IgePal-630, 0.5% deoxycholate, 1 mM EDTA) containing a protease inhibitor mixture (Cocktail Set III) and phosphatase inhibitors (Cocktail Set V) (EMD Millipore, Billerica, MA). Following centrifugation, whole cell supernatants were collected and stored at -80˚C. Protein concentration was measured using the BCA assay (Pierce). Equal protein amounts (4–6 μg) from the stimulated HLEC lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using NuPAGE 4–12% Bis-Tris or 3–8% Tris-Acetate mini-gels (Invitrogen) under denaturing conditions. The separated proteins were electroblotted onto polyvinylidene difluoride membranes (pore size, 0.45 μm; Invitrogen). Protein blots were blocked for 1 h in TBS containing 0.1% Tween 20 (TBST) with 5% nonfat dry milk or 5% BSA and probed with the specific primary antibody followed by HRP-conjugated secondary antibody. Immunodetection was revealed on HyperECL film with the ECL Plus chemiluminescence detection system (GE Healthcare UK Ltd., Buckinghamshire, United Kingdom) according to the manufacturer's instructions. The relative amount of protein was estimated by densitometry analysis with Image J software (National Institutes of Health, Bethesda, MD).