Western Blot Analysis for the Detection of Cleaved Caspase-3

Protein expressions for cleaved caspase-3 were measured using Western blotting technique (Nemmar et al., 2018). Kidney tissues harvested from the mice were snap frozen immediately with liquid nitrogen and stored at −80°C. Later, the tissues were weighed, rinsed with saline and homogenized with lysis buffer (pH 7.4) containing NaCl (140 mM), KCl (300 mM), trizma base (10 mM), EDTA (1 mM), Triton X-100 0.5% (v/v), sodium deoxycholate 0.5% (w/v), protease, and phosphatase inhibitor. The homogenates were centrifuged for 20 min at 4°C. The supernatants were collected and protein estimation was made using a Pierce bicinchoninic acid protein assay kit (Thermo Scientific). A 35 μg sample of protein was electrophoretically separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes. The immunoblots were then blocked with 5% non-fat milk and subsequently probed with rabbit monoclonal cleaved caspase-3 antibody (1:250 dilution, Cell Signalling Technology) at 4°C overnight. The blots were then incubated with goat anti-rabbit IgG horseradish peroxidase conjugated secondary antibody (1:5000 dilution, Abcam) for 2 h at room temperature and developed using Pierce enhanced chemiluminescent plus Western blotting substrate Kit (Thermo Scientific). The densitometric analysis of the protein bands was performed with Typhoon FLA 9500 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Blots were then re-probed with mouse monoclonal GAPDH antibody (1:5000 dilution, Abcam) and used as a control.