Rabbit anti perk1 2 antibody

24h after transfection, HEK 293T cells were starved in Waymouth/BSA at 37°C for 24h. After treated with or without 1 μM NDP-MSH for 5 min at 37°C on the second day, the cells were transferred directly on ice, washed twice using cold 0G (150 mM NaCl and 20 mM Hepes, pH 7.4), and then scraped into lysis buffer (0G containing 0.5% NP-40, 2 mM EDTA, 1 mM Na₃VO₄, and 1 mM NaF). Total protein concentrations were determined by Bradford protein assay and 35 μg protein samples were separated on 10% SDA-PAGE gel and then blotted onto PVDF membranes. After blocking in 10% nonfat dry milk (containing 0.2% Tween-20) for at least 4 h at room temperature, the membranes were then immunoblotted with rabbit anti-pERK1/2 antibody (Cell Signaling, Beverly, MA, USA) 1:2000 and mouse anti-β-tubulin antibody (Developmental Studies Hybridoma Bank, The University of Iowa, Iowa City, IA, USA) 1:5000 diluted in Tris-buffered saline containing Tween 20 (TBST) with 5% BSA overnight at 4 °C. The membranes were then probed with HRP-conjugated secondary antibody, donkey anti-rabbit (Jackson ImmunoResearch, West Grove, PA, USA) 1:1500 and donkey anti-mouse IgG (Jackson ImmunoResearch) 1:5000 diluted in 10% nonfat dry milk for 2 h at room temperature. Specific bands were visualized using ECL reagent (Thermo Scientific, Rockford, IL, USA) and then analyzed using Image J software (NIH, Bethesda, MD, USA).