Superoxide dismutase 1

Cells were lysed on ice for 15 min in either radioimmunoprecipitation assay lysis buffer (Boston BioProducts) or EBC lysis buffer (50 mM Tris [pH 8.0], 250 mM NaCl, and 0.5% Nonidet-P40 [NP-40]) containing phosphatase and complete protease inhibitors (Roche). Lysates were cleared via centrifugation and quantified using the Bradford Reagent (Bio-Rad Laboratories). Lysates were mixed with 2× sample buffer (0.125 M Tris [pH 6.8], 4% SDS, and 20% glycerol) containing 4% β-mercaptoethanol and boiled for 5 min. Nuclear cytoplasmic fractionations were performed using the Nuclear Extract Kit (Active Motif) according to the manufacturer's protocol. Samples were resolved on 10 to 12% acrylamide gels and transferred to 0.45 μm Amersham Protran nitrocellulose (GE Healthcare). Membranes were blocked with 5% milk in TBS/Tween (Boston BioProducts) and probed with antibodies to pSTAT3 (catalog no.: 9131; Cell Signaling), STAT3 (catalog no.: 482, Santa Cruz Biotechnology; catalog no.: 9139, Cell Signaling), DHFR (catalog no.: 377091; Santa Cruz Biotechnology), tubulin (catalog no.: T5168; Sigma–Aldrich), and superoxide dismutase 1 (catalog no.: 11407; Santa Cruz Biotechnology). Immunoblots were developed, and band intensity was quantified using Fiji (42 (link)).

Tissues were lysed in urea buffer (7M urea, 2M thiourea and 4% CHAPS) containing protease inhibitor cocktail and the concentration of the protein was determined by the standard Bradford method. Equal amount of proteins (40 μg) was separated on 12–15% SDS-PAGE and transferred to PVDF membrane. After transfer, gel profile on the membrane was visualised with Ponceau Sto check equal loading [15 (link)]. Membranes were blocked using 3% BSA and incubated with Superoxide dismutase 1 (Santa Cruz Biotechnology Inc., USA), cytoplasmic 1 actin (Sigma Aldrich, St. Louis MO, USA) at room temperature for2 hrs. Subsequently, membranes were washed with PBS—0.1% Tween-20 (PBST) thrice for 5 minutes and were then incubated with their secondary antibodies conjugated with HRP for 2 hrs at room temperature. The membrane was washed thrice for 5 minutes with PBST and protein bands were visualised by enhanced chemiluminescence (ECL, Amersham Biosciences, Bucks, UK) in UVP Bio-Spectrum Imaging System and the densitometry analysis was performed with Image J software (http://rsbweb.nih.gov/ij/).