Anti erk sc94

Proteins were extracted and visualized as reported in [30 (link)]. Immunoblotting was carried out with the following antibodies: anti-p70S6K(Thr389) #9206, anti-p70S6K #9202, anti-S6(Ser235/236) ribosomal protein #2211, anti-S6 ribosomal protein 2217#, anti-4E-BP1(Thr37/46) #2855, anti-4E-BP1 #9644 provided by Cell Signalling Technology. Anti-Erk #sc94, anti-Erk(Tyr204) #sc7383, anti-KRAS #sc30 and anti-tubulin#9104 were obtained from Santa Cruz Biotechnology. Anti-LC3 #PM036 was obtained from MBL.

Cellular protein extracts from cell lines were prepared using a cell lysis buffer containing 18 mmol/L Tris-HCl, pH 7.5; 135 mmol/L NaCl, 0.9 mmol/L EDTA; 0.9 mmol/L EGTA and supplemented with 1 mmol/L PMSF; 1× EDTA-free cocktail protease inhibitor (Roche Diagnostics, Bale, SWISS); 30 mmol/L sodium fluoride, 40 mmol/L glycero phosphate; 1 mmol/L sodium orthovanadate; and 0.5% Triton X-100.
Protein concentrations were determined using the BCA protein assay (Sigma-Aldrich, St. Louis, MO, USA) with bovine serum albumin as a standard. Protein samples were denatured for 10 min at 95 °C, and equal amounts of cell proteins (50 μg) were subjected to 10% SDS-PAGE and transferred onto nitrocellulose membranes (Amersham-GEH Life, Buckinghamshire, UK). The membranes were probed with the appropriate antibodies. The primary antibodies used were: anti-CDK4 (559693, BD Pharmingen, San Diego, CA, USA), anti-MDM2 (clone 2A10, ref MABE281, Merckmillipore, Darmstadt, Germany), and anti–ERK (sc-94, Santa Cruz Biotechnology, Dallas, TX, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Signals were detected using a LAS-4000 Imager (Fuji Photo Film, Tokyo, Japan).