Peroxidase conjugated goat anti rabbit immunoglobulin

The kidneys were homogenized in chilled RIPA lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton x-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 2 mM EDTA, and 50 mM NaF) at a ratio of 100 mg of tissue to 1 ml of buffer. A protease inhibitor cocktail (Pierce, Thermo Fisher Scientific, Waltham, MA, USA) and sodium orthovanadate were added to the lysis buffer prior to use. Lysates were incubated at 4°C for 20 min, and then centrifuged at 15,000 x g, 4°C, for 20 min. Protein concentration was determined by the BCA Protein Assay Kit (Pierce, Thermo Fisher Scientific) and samples were stored at -70°C until analysis.
For Western blotting, equal amounts of protein samples were run on 10% polyacrylamide gels and transferred to nitrocellulose membranes (Appli-Chem GmbH, Darmstadt, Germany). Membranes were probed with primary antibodies to Nox2 (1:500, Abcam, ab31092), and actin (1:1000, Sigma-Aldrich, A5060). Peroxidase-conjugated goat anti-rabbit immunoglobulin (1:40.000, Sigma-Aldrich, A0545) was used as a secondary antibody. Western blots were developed using the enhanced chemiluminescence reagent system (GE Healthcare, Amersham, UK) according to the manufacturer’s instructions. The content of Nox2 in the tissue extracts was estimated by the densitometry of scanned immunoblot bands using the Image Master Total Lab (GE Healthcare) software.

The renal tissues were homogenized in lysis buffer, as previously described [59 (link)]. Equal amounts of protein samples (concentration determined by BCA assay, Thermo Fisher scientific, USA) were separated by 12% SDS-PAGE and transferred to nitrocellulose membrane (AppliChem, Darmstadt, Germany). Next, membranes were blocked with 5% nonfat milk in TBS-Tween-20 (Serva, Heidelberg, Germany) for 1 h and probed with appropriate dilutions of primary antibodies for Bax (1:1000, 04-434 Millipore, Burlington, MA, USA), Bcl-2 (1:250, 05-729 Millipore), HO-1 (1:250, ab13248 Abcam, Cambridge, UK), and actin (1:500, A5060 Sigma-Aldrich) overnight at 4 °C. Peroxidase-conjugated goat anti-rabbit immunoglobulin (1:40,000, A0545 Sigma-Aldrich) or goat anti-mouse immunoglobulin (1:20,000, A5278 Sigma-Aldrich) were used as secondary antibody. Enhanced chemiluminescence (ECL) method (Serva, Heidelberg, Germany) and quantified using Image Lab software (Bio-Rad) were used for protein bands visualization.