Phospho p44 42 mapk erk1 2 4370

Cells were lysed in Laemmli buffer 1x (Tris 60 μM pH 6.8, 2% SDS, 10% glycerol, and 5% 2-β mercaptoethanol). Equal volumes of extracts were separated by SDS-PAGE and transferred to nitrocellulose membranes (Millipore, MA, USA). Blots were blocked for 1 hour in 5% nonfat milk (Bio-Rad Laboratories, Inc., Hercules, CA, USA) prepared in TBST 1x and incubated overnight with the following primary antibodies: α-ERK1 (K23; 1 : 1000) and α-CDK4 (c-22; 1 : 1000) (Santa Cruz Biotechnology, Inc., CA, USA), phospho-p44/42 MAPK ERK1/2 (4370; 1 : 1000) (Cell Signalling Technology Boston, USA), and α-Vimentin (Clone V9; 1 : 1000) (Millipore, MA, USA). Blots were incubated with HRP-conjugated anti-rabbit secondary antibody (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Immunoreactivity was detected by Enhanced Chemiluminescence Reaction (WESTAR NOVA 2011, Cyanagen, Bologna, Italy) following the manufacturer's instructions.

Antibodies were obtained from the different sources. PKC‐δ (2058), phospho‐PKC‐δ (9374), Caspase3 (9662), cleaved caspase3 (9664), p38 (8690), phospho‐p38 (4511) and phospho‐p44/42 MAPK (Erk1/2) (4370) were from Cell Signaling Technology (Danvers, MA, USA); anti‐Erk1/2 (ab184699) from Abcam (Cambridge, UK); β‐Tubulin (T0023) were from affinity (UK); Myoglobin and ascorbic acid were from Sigma‐Aldrich (St. Louis, MO, USA). Rottlerin (the inhibitor of PKC) were from Abcam (Cambridge, UK);