Immunoblotting for p38 MAPK and phospho-p38 MAPK

Renal cortex tissue was placed into ice-cold 1% Triton lysis buffer (containing a protease inhibitor cocktail (Sigma-Aldrich Chemie GmbH)) and immediately homogenized. Lysates were centrifuged at 20,500 g for 10 min at 4 ºC. We measured protein concentrations using a Bradford assay (BioAssay Systems, Hayward, USA). After dithiothreitol treatment (100 mM) and denaturation (5 min at 95 °C), 30 µg of total protein were loaded onto 10% SDS-PAGE gels and then transferred to nitrocellulose membranes, according to the manufacturer’s instructions (X-Cell Blot Module, Invitrogen / Thermo Fisher Scientific Inc., Oberhausen, Germany). Membranes were treated with blocking buffer (5% bovine serum albumin (BSA) and 0.1% Tween 20 in phosphate buffered saline (PBS)) for 1 h at room temperature, and then incubated either with primary monoclonal rabbit anti-p38 antibody at 1:750 (Cell Signaling Technology, Europe, Leiden, The Netherlands) or primary monoclonal rabbit anti-phospho-p38 antibody at 1:1300 (Cell Signaling Technology), and then mouse anti-β-actin at 1:20,000 (Santa Cruz Biotechnology, Inc., Dallas, USA) overnight. Bound primary antibody was detected with anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody at 1:10000 (Dako Deutschland GmbH, Hamburg, Germany) by 60 min incubation at room temperature. Antibody labeling was visualized by the addition of a chemiluminescence reagent (Lumi-LightPLUS Western Blotting Substrate, Sigma-Aldrich Chemie GmbH). Chemiluminescence was visualized using a FluorChem FC2 Imager (alpha innotec / Protein Simple, San Jose, USA). Immunoblots from each tissue were performed in triplicate.