Anti p p38 thr180 tyr182

Fresh oocytes were lysed by pippeting up and down in 200 μl (pools of 20 oocytes) of ice-cold extraction buffer (0.25 M sucrose, 0.1 M NaCl, 2.5 mM MgCl₂, 20 mM HEPES, pH 7.2) containing 1 mM EDTA, 1 mM EGTA, protease inhibitors (10 μg/ml leupeptin, 1 mM PMSF, 10 μg/ml aprotinin) and phosphatase inhibitors (50 mM β-glycerolphosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 5 mM sodium pyrophosphate). Samples were clarified by centrifugation at 14,500 rpm for 5 min and supernatants were collected and processed for immunoblotting or caspase assay as described below. The whole supernatants were denatured with Sample Buffer (50 mM Tris HCl, pH 6.8, SDS 2%, 100 mM dithiothreitol, 10% glycerol) and subjected to 10% or 15% SDS/PAGE and transferred to Immobilon-P membranes (Millipore). Uniformity of samples loading was verified by Ponceau (Sigma) staining of the blots. Membranes were blocked for 1 h with 5% dried skimmed milk in TBST (50 mM Tris, 150 mM NaCl, 100 mM KCl, pH 7.4, and 0.1% Tween 20) and then incubated with the following antibodies: anti-AMPKα (#2532, Cell Signaling), anti-pp38 (Thr180/Tyr182) (#9211, Cell Signaling), anti-p38 (sc-7149, Santa Cruz), and anti-Cytochrome c (556432, BD Pharmingen). Antibody binding was detected with horseradish peroxidase–coupled secondary antibody and the enhanced chemiluminescence (ECL) detection kit (Amersham).

Isoproterenol and Trichostatin A (TSA) were purchased from Sigma Aldrich (St. Louis, MO, USA) and used at 10 µM and 100 nM, respectively. Murine TNF-α was a gift from the VIB Department for Molecular Biomedical Research of Ghent University (VIB-UGent, Gent, Belgium) and was used at 2000 IU/ml. Insulin-like growth factor-1 (IGF-1) was from ImmunoTools (Friesoythe, Germany) and was used at 10 ng/ml. Anti-β₂-AR, anti-TNF-R1, anti-myogenin, anti-PARP, anti-P-H3-Ser10, anti-CBP, anti-RNA polymerase II, anti-p65, anti-IκBα, anti-P-CREB-Ser133 and anti-PKAc were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-P-p65-Ser536, anti-P-ERK-Thr202/Tyr204, anti-P-JNK-Thr183/Tyr185, anti-P-p38-Thr180/Tyr182, anti-P-MSK-1-Thr581, anti-Lamin A/C and anti-CREB were from Cell Signaling Technology (Danvers, MA, USA). Anti-Ac-H3-Lys27 and GAPDH were from AbCam (Cambridge, UK). Anti-α-tubulin and anti-α-actin were from Sigma-Aldrich. In figures, the expression “antibody anti-” was substituted by the Greek letter “α-”. AatII and HincII restriction enzymes were obtained from New England BioLabs (Ipswich, MA, USA).