Rabbit anti mkk4

Cells were lysed in RIPA buffer (50 mMTris–HCl, 150 mM NaCl, 1 % NP-40, 0.5 % sodium deoxycholate, 0.1 % SDS, PH 8.0) supplemented with a protease inhibitor cocktail (Roche Applied Science). The protein concentration was measured using a Bradford protein assay kit (Coomassie Plus Protein Assay reagent, Thermo). Protein samples were separated by 10 % SDS-PAGE (Bio-Rad) and electroblotted onto 0.45 µm Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked with 5 % Blotting-Grade Blocker (Bio-Rad) in PBST for 1 h at room temperature and incubated overnight at 4 °C with the respective antibodies: rabbit anti-MKK4, rabbit anti-JNK1, rabbit anti-phosphate JNK1, and rabbit anti-phosphate c-Jun (1:1,000; Cell Signaling, Danvers, MA); mouse anti-GAPDH antibody (1:4,000; Millipore, Billerica, MA); or rabbit anti-cleaved caspase 3 (1:500, Sigma-Aldrich, St. Louis, MO). Then, membranes were incubated for 1 h at room temperature with Amersham ECL peroxidase-linked secondary antibodies: sheep anti-mouse IgG (1:10,000, GE Healthcare) or donkey anti-rabbit IgG (1:10,000, GE Healthcare). Western blot immunoreactivity was detected using a Super Signal West Femto Maximum Sensitivity Substrate Kit (Thermo) with a C-DiGit Blot Scanner (LICOR Biosciences, Lincoln, Nebraska).