Donkey anti goat antibodies

ADPKD cells were seeded onto T25 culture flasks at a density of 2.5 × 10⁵ cells/flask. The cells were incubated with test agents depending on the experiments. After treatment, cells were lysed with Triton X-100 lysis buffer containing protease inhibitors. The supernatant from whole-cell lysates was collected by centrifugation. For p53 activation, nuclear and cytoplasmic proteins were separated using the NucBuster Protein Extraction Kit (Novagen, Rockland, USA). Protein concentrations were determined using the Bradford assay. Later, 30 μg total protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated protein bands were blotted onto polyvinylidene fluoride membranes (Bio-Rad). The membranes were blocked with 5% bovine serum albumin and then probed with primary antibodies for p-MEK-1/2, p-ERK-1/2, ERK-1/2, p53, caspase-9, caspase-8, caspase-3, or β-actin. The protein of interest was detected by incubation with secondary horseradish peroxidase-conjugated goat anti-mouse (Santa Cruz Biotechnology) or donkey anti-goat antibodies (Santa Cruz Biotechnology) and visualized with an enhanced chemiluminescence solution from GE Healthy Care Life Science using a ChemiDoc MP system (Bio-Rad). β-Actin was used as a control.