Anti p p44 42 mapk erk1 2 thr202 tyr204

Cells were collected and lysed with NP-40 lysis buffer with protease inhibitors. Lysates were analyzed for protein expression/phosphorylation as described previously [25 (link),27 (link)]. The following antibodies were used at the indicated dilutions; all antibodies were purchased from Cell Signaling (Danvers, MA, USA): anti-p-AKT-S473 (1:1000, #4060), anti-AKT (1:1000, #4685), anti-p-p44/42 MAPK (Erk1/2) -Thr202/Tyr204 (1:1000, #4370), anti- p44/42 MAPK (Erk1/2) (1:1000, #4695), anti-p-p38 MAPK-Thr180/Tyr182 (1:1000, #9211), anti-p38 MAPK (1:1000, #8690), anti-p-cdc2-Tyr15 (1:1000, #4539), anti-cdc2 (1:1000, #9116), anti-p-CDC25C-Ser216 (1:1000, #4901), anti-cdc25C(1:1000, #4688), anti-p-Histone H3-ser10 (1:1000, #53348).

Liver tissue was homogenized in HEPES buffer and proteins were separated by SDS-PAGE and transferred to PVDF membranes. Then, membranes were immunoblotted over night at 4°C using the following antibodies: anti-p-AMPKα (Thr172, #2535), anti-AMPKα (#2603), anti-p-SAPK/JNK (Thr183/Tyr185), anti-p-p38 MAP kinase (Thr180/Tyr182, #9211), and anti-p-p44/42 MAPK (Erk1/2, Thr202/Tyr204, #9101); the above antibodies were all purchased from Cell Signaling (Danvers, MA); anti-eNOS (610296) was purshased form Transduction Laboratories (Lexington KY) and anti-b-actin (A5316) was purshased from Sigma Chemical (St. Louis, MO, USA). After washing, bound antibody was detected after incubation for 1 h at room temperature with the corresponding secondary antibody linked to horseradish peroxidase. Bound complexes were detected and quantified by scanning densitometry.