Anti phospho sapk jnk thr183 tyr185 antibody

We lysed 100 mg pancreas tissues using radioimmunoprecipitation assay buffer with 1% PMSF on ice. The total protein concentration was determined using a Bradford protein kit (Bio-Rad, Richmond, CA, USA). The proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto Immobilon-P Transfer Membranes (Millipore Japan, Tokyo, Japan). The membranes were probed with primary antibodies for 2 h at room temperature. The primary antibodies used were anti-Phospho-SAPK/JNK (Thr183/Tyr185) antibody (1: 1000, Cell Signaling Technology, 4668), anti-SAPK/JNK (1: 1000, Cell Signaling Technology, 9252), anti-p-IκBα antibody (1: 500, Santa Cruz Biotechnology), anti- IκBα antibody (1: 5000, Cell Signaling Technology), anti-PAI-1 antibody (1: 1000, Abcam, ab66705), and anti-TNF-a antibody (1: 1000, Abcam, ab1793). The membranes were incubated for 1 h with secondary antibody at room temperature (1: 2000, Cell Signaling Technology). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1: 2500, Abcam, ab9485) was used as an internal control. Antigen–antibody complexes were then visualized using an electrochemiluminescence kit (GE Healthcare). The protein levels were normalized against those of GAPDH by using Image J software (National Institute of Health, Bethesda, MD, USA). All experiments were repeated in triplicate.