SOD-2, PPARα and γ expression markers were assayed through western blotting analyses. The proteins have been extracted using Radio-Immunoprecipitation Assay (RIPA) buffer R0278 (Sigma Aldrich, Milan, Italy) and the concentrations were determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Milan Italy). Equal amounts of protein (30 μg) were loaded on a SDS-PAGE and transferred to a nitrocellulose membrane [19 –22 (link)]. The filters were incubated with a monoclonal antibody against SOD-2 (A-2, sc-133,134) diluted 1:250 v/v, polyclonal antibody against PPARα (H-98, sc-9000) diluted 1:200 v/v, monoclonal antibody against PPARγ (E-8, sc-7273) diluted 1:250 v/v, polyclonal antibody against Actin (I-19, sc-1616) diluted 1:500 v/v, at room temperature for 2 h. All reagents were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Membranes were washed three times for 10 min and incubated with a 1:10,000 v/v dilution of horseradish peroxidase-conjugated anti-mouse, anti-rabbit and anti-goat antibodies for 1 h respectively. Blots were developed using ECL system (Amersham Biosciences, UK) according to the manufacturer’s protocols.
Free full text: Click here
