Manganese superoxide dismutase mnsod

Funato lysis buffer (50mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA pH 8, 1% Triton X-100, Roche protease inhibitor) was used to aid homogenisation of kidney cortex by a TissueRuptor (Qiagen, Hilden, Germany). The resulting homogenate was centrifuged at 10,000g for 15 min and the supernatant protein concentration was determined with the Pierce BCA Protein Kit (Thermo Fisher Scientific, VIC, Australia). 20 μg of protein was loaded onto an Invitrogen Bolt 4–12% Bis-Tris Plus Gel (Thermo Fisher Scientific) for electrophoresis (n = 6), before transfer to a Hybond Nitrocellulose membrane (Amersham Pharmacia Biotech, Bucks, UK). Incubation of membranes at 4°C overnight was performed using the following primary antibodies: fibronectin (dilution 1:2000, Thermo Fisher Scientific), collagen III (1:2500, Abcam, Cambridge, UK), collagen IV (1:5000, Abcam), manganese superoxide dismutase (MnSOD 1:2000, Merck Millipore Ltd, Darmstadt, Germany), and α-tubulin (1:2000, Sigma-Aldrich, MO, USA). Membranes then underwent incubation with a horseradish peroxidase-conjugated secondary antibody, and were developed using ImageQuant LAS 4000 (Fujifilm, Tokyo, Japan). GelPro Analyser software (Informer Technologies Inc.) was used for analysis. Protein expression was expressed as a proportion relative to α-tubulin, a commonly used internal control.