Jnk sc 7345

Cells were collected and lysed with radio immunoprecipitation assay buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and protease/phosphatase inhibitor cocktail). Total cell lysate concentrations were determined using a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins (50 µg/ml) were separated by SDS-PAGE (10% running and 4% stacking). Equal amounts of protein from the control and experimental groups was suspended in 5X sample buffer and distilled water and boiled for 10 min, then subjected to SDS-PAGE. The electrophoresis was carried out for 2 h and the separated proteins were transferred to a nitrocellulose membrane. Following blocking with 5% skim milk for 1 h at room temperature, the membrane was blotted with the JNK-(sc-7345) and phosphor-JNK (sc-6254; both Santa Cruz Biotechnology) specific primary antibody (diluted in 1:500) overnight at 4°C. The membranes were washed with Tris-buffered saline with Tween-20 buffer, they were blotted with anti-mouse secondary antibody (1:3,000; cat. no. 7076; Cell Signaling Technology, Inc., Danvers, MA, USA) for 2 h at room temperature. Immunodetection was performed by using a western light chemiluminescent detection system (Applied Biosystems; Thermo Fisher Scientific Inc.).