Mouse monoclonal anti phospho p44 42 mapk erk1 2 thr202 tyr204

Protein extracts were prepared from tissue samples (∼50 mg) by homogenization in 500 μl whole cell lysis buffer (1% NP-40, 10 mM Tris pH 7.8, 150 mM NaCl, 40 mM EDTA, 10 mM Na-Pyrophosphate, 10 mM NaF, 1mM PMSF, 4 mM Orthovanadate, Minicomplete iprotease inhibitor^®). Protein extracts (30 μg) were separated by polyacrylamide gel electrophoresis. Blottings were incubated one hour at RT in a blocking buffer containing 5% skim milk and then incubated overnight at 4°C with mouse monoclonal anti-phosphorylated STAT3 (Santa Cruz), rabbit monoclonal anti phosphorylated AKT (Ser473) (Cell Signaling, Danvers, MA), mouse monoclonal anti Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling, Danvers, MA), mouse monoclonal anti Diphosphorylated-JNK (JNK-PT48) (Sigma) and beta-actin mouse monoclonal antibody (Sigma-Aldrich), and subsequently, with peroxidase-conjugated goat anti-mouse or anti-rabbit immunoglobulin G (Dako) for one hour at room temperature.

Protein contents in whole-cell lysates were resolved using 8–12% SDS-PAGE and subsequently transferred to polyvinylidene fluoride membranes (Perkin Elmer, Santa Clara, CA, USA). Western blotting was performed [67 (link)] using antibodies obtained from the following sources: rabbit monoclonal anti-class III PI3K, anti-phospho-Aurora-A (Thr288), anti-phospho-MEK1/2 (Ser221)(166F8), anti-phospho-RB (Ser780), rabbit polyclonal anticaspase 3, anti-phospho-mTOR (Ser2448), anti-class I phospho-PI3K p85 (Tyr458)/p55 (Tyr199), mouse monoclonal anti-phospho-p44/42 MAPK (ERK 1/2) (Thr202/Tyr204), and anti-phospho-p70S6K (Thr389) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA); rabbit polyclonal anti-LC3B and rabbit monoclonal anti-APG5L/Atg5 were obtained from Abcam (Cambridge Science Park, UK); mouse monoclonal anti-pan-RAS was obtained from Calbiochem (San Diego, CA, USA); and mouse monoclonal anti-β-actin was obtained from Sigma; goat antirabbit and antimouse conjugated horseradish peroxidase secondary antibodies were obtained from Millipore Corp. (Billerica, MA, USA). The whole western blots (uncropped blots) could see Figure S9.