Anti phospho c jun n terminal kinase

The cytoplasm and nuclear fraction of cells was extracted with the ProteoExtract Subcellular Proteome Extraction Kit (Calbiochem, San Diego, CA, USA). The protein content in the cell lysates was determined using a BCA protein-assay kit. The extracts (40 μg of protein) were fractionated on polyacrylamide-SDS gels and transferred to PVDF membranes (GE Healthcare, Buckinghamshire, UK). The membranes were blocked with a solution containing 3% skim milk and incubated overnight at 4 °C with each of the following antibodies: anti-NF-κB p65, anti-phospho-ERK1/2 antibody, anti-phospho-Akt antibody, anti-phospho-mammalian target of rapamycin (mTOR) antibody, anti-phospho-p38MAPK, antiphospho-c-Jun N-terminal kinase (JNK), antibody, anti-ERK1/2 antibody, anti-Akt antibody, anti-mTOR antibody, anti-p38MAPK antibody, anti-HIF-1α antibody (Cell Signaling Technology, Beverly, MA, USA), anti-MDR antibody, anti-Survivin antibody, anti-Bim antibody, anti-lamin A/C antibody (Santa Cruz Biotechnologies, CA, USA), and anti-β-actin antibody (Sigma). Subsequently, the membranes were incubated with horseradish peroxidase-coupled anti-rabbit IgG sheep antibodies (GE Healthcare) for 1 h at room temperature. The reactive proteins were visualized using Luminata Forte (Merck Millipore, Nottingham, UK) according to the manufacturer's instructions.