Radio immunoprecipitation assay buffer ripa buffer

The protein was extracted using Radio-Immunoprecipitation Assay Buffer (RIPA buffer, Santa Cruz, CA) and the protein concentration was determined using the BCA^TM Protein Assay Kit (Pierced, Grand Island, NY). Samples were separated by electrophoresis on 10-12% sodium dodecyl sulfate (SDS) polyacrylamide gels, and the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). To assess the protein expression, the blots were incubated with the following primary antibodies at 4°C overnight: rabbit antibodies against βTrCP, ZO-1, E-cadherin, ZEB1, N-cadherin, Vimentin, and Slug (Cell Signaling Technology), as well as mouse antibodies against Snail (Cell Signaling Technology), and β-catenin (BD Biosciences, New Jersey). After washing, the blots were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (Cell Signaling Technology) at a dilution of 1:2000 for 1 h at room temperature. Blots were visualized by exposure to X-ray film, through an enhanced chemiluminescence detection system (Millipore). GAPDH (Cell Signaling Technology) served as an endogenous control for equal loading.

Monosodium glutamate (SRL, India), 5,59-dithio-bis (2-nitro benzoic acid) (DTNB), Triton X-100, Trichloroacetic acid (TCA), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Thiobarbituric acid (TBA), Tween 20 and Bovine serum albumin (BSA) were purchased from Sigma Aldrich (USA). Antibodies such as Bcl2, Bax, p21, p53, NF-kB (p65), cleaved caspase 3, cleaved caspase 9 and GAPDH, 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Cell Signalling Technology (USA) and PPAR-α from GeneTex (USA), PPAR-γ from Abcam (USA). Ethidium bromide (EtBr), ethanol, hydrogen peroxide (H₂O₂), petroleum ether, chloroform, n-hexane, acetone and all other chemicals were procured from Merck (Germany). Anti-rabbit IgG fluorescein isothiocyanate (FITC) and Radioimmunoprecipitation assay buffer (RIPA) buffer were procured from Santa Cruz Biotechnology (USA). 3-3′-dihexyloxacarbocyanine iodide (DiOC6, Molecular Probes), 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and RNase-PI were procured from Thermo Fisher Scientific (USA). All the cell culture media, buffer, collagenase type II, Trizol reagent and other reagents were obtained from Gibco (USA) and all other reagents used for this study were of highest quality grade.