Anti p44 42 mapk total erk

Samples were lysed in NP40 lysis buffer (Lifetechnologies) containing protease inhibitor set I (Millipore), 5 mmol/L orthovanadate, and 2 mmol/L sodium pyrophosphate, then briefly sonicated, incubated for 15 minutes at 4°C and centrifuged 15 minutes at 4°C at 13 000 rpm. Supernatants were immediately frozen.
Proteins (2.5 μg/sample) were separated using SDS-PAGE (NOVEX Nupage 4% to 12%; LifeTechnologies) and transferred to a nitrocellulose membrane (Amersham Protran Premium 0.45 NC; GE Healthcare Lifesciences). All gels and transfers were run in double; one membrane was incubated with anti-Phospho-p44/42 MAPK (p-ERK, Cell Signaling 4370) and the other with anti-p44/42 MAPK (total ERK, Cell Signaling 9102). Membranes were blocked in TBST containing 5% nonfat dry milk for 1 hour at room temperature and then incubated overnight at 4°C with primary antibody diluted in blocking buffer (1:1000). Membranes were washed in tris buffer saline-tween 20 (TBST) and then incubated with horseradish peroxydase (HRP)-conjugated secondary antibody (1:1000 in blocking buffer, Pierce, 31460). Bands were developed using Supersignal west Femto substrate (ThermoFisher). Membranes were imaged and bands quantified using the GE Healthcare ImageQuant LAS-4000 system.