Phospho erk1 2 and erk1 2 antibodies

Cells were lysed in a buffer containing 50 mM Tris (pH 7.9), 150 mM NaCl, 1% NP40, 1 mM EDTA, 10% glycerol, 1 mM sodium vanadate, and protease inhibitor cocktail (Roche Pharmaceuticals, Nutley, NJ). Proteins were separated by SDS-PAGE with 4–20% gradient gels (Bio-Rad Laboratories, Hercules, CA), transferred to a Hybond-ECL nitrocellulose membrane (GE Healthcare Biosciences, Piscataway, NJ), and blocked in 5% dry milk in PBS. The membrane was then incubated with primary and secondary antibodies, and target proteins were detected with ECL detection reagent (GE Healthcare Biosciences).
MET(C-12), PARP and beta actin antibodies were obtained from Santa Cruz Biotechnology, Phospho-Met antibody was purchased from Invitrogen, Phospho Erk1/2 and Erk1/2 antibodies were obtained from Cell Signaling Technology.

Equal amounts of proteins (20 μg), as determined by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA), were resolved on a 8% or on a 12% SDS-PAGE and electrotransferred to Amersham Hybond PVDF membrane (GE Healthcare, Milan, Italy). Membranes were then blocked for 1 h at 25 °C in PBS containing 5% nonfat dry milk and 0.1% Tween-20 (Sigma-Aldrich), and were incubated for 1 h at 25 °C or overnight at 4 °C, with the following primary antibodies: BRLF1 and BZLF1(1 : 200 and 1 : 100, respectively, both obtained from Argene Biosoft, Verniolle, France), BALF5 (1 : 100), LC3 (1 : 8000, L7543) and β-actin (1 : 5000) were purchased from Sigma, Beclin1(1 : 500; sc-10086) and ATG5 (1 : 200, sc-133158) purchased from Santa Cruz (DBA, Milan, Italy), and phospho-p70 S6 kinase (1 : 1000, 07-018-I Millipore, Merk Spa, Vimodrone, MI, Italy). Phospho ERK1/2 and ERK1/2 antibodies (1 : 500) were obtained from Cell Signaling (EuroClone, Milan, Italy). The membranes were then incubated with the appropriate secondary antibodies conjugated to HRP (1 : 7500, Bio-Rad). The specific signals, visualized by Amersham ECL Plus kit (GE Healthcare) were quantified by densitometric analysis by ImageJ free-share software (http://imagej.nih.gov/ij).