Ecl super signal kit

Equal amount of protein from each sample was separated in 10% SDS-PAGE and transferred to the PVDF membrane overnight at 4°C. Membrane was blocked in 5% non-fat milk in PBS (pH 7.4) for 2h at RT. Blot was incubated overnight at 4°C with rabbit anti-mouse p53 (1:500 dilution, Imgenix) or rabbit anti-mouse iNOS (1:200 dilution, Cayman) or rabbit anti-mouse COX2 (1:500 dilution, Cayman) in 1% BSA and 0.05% Tween-20 in PBS (pH 7.4). Blot was washed and incubated with HRP-conjugated goat anti-rabbit IgG (1:2500 dilution, Bangalore Genei) in PBS (pH 7.4) containing 1% BSA and 0.05% Tween-20 for 2 h at RT. Immunoreactive proteins were detected with ECL super signal kit (Pierce Biotechnology) in X-ray film. Band density values were normalized with β-actin.

Catalytic activity of PKCδ and caspase-3 were determined by Western blot analysis. Equal amount of protein from each sample was separated in 10 % SDS–PAGE and transferred to the PVDF membrane overnight at 4 °C. Membrane was blocked in 5 % non-fat milk in PBS (pH 7.4) for 2 h ar RT. The blot was then incubated with rabbit anti- caspase-3(Product number C8487) or rabbit anti- PKCδ (AB-645; Product number SAB-4300539) in 1 % BSA and 0.05 % Tween-20 in PBS (pH 7.4). Blot was washed and incubated with HRP-conjugated goat anti-rabbit IgG in PBS (pH 7.4) containing 1 % BSA and 0.05 % Tween-20 for 2 h at RT. Immunoreactive proteins were detected with ECL super signal kit (Pierce Biotechnology) in X-ray film. The same blot was used for the detection of β-actin as internal control. Band density values were normalized with β-actin.

All chemicals were of analytical and molecular biology grade as well as endotoxin free and used without further purification. Curcumin, reagents for RNA isolation and Taq polymerase, Nonfluorescent 2′, 7′-dichlorofluorescein diacetate (H2DCFDA) and HRP conjugated anti-β actin were purchased from Sigma Aldrich. Reverse Transcriptase, Ribonuclease Inhibitor, random primers and 100 bp Plus DNA ladder from Fermentase Life Science and gene specific primers for RT-PCR were synthesized from Metabion. Anti-mouse PKCα and VEGF-A antibody were purchased from Santacruz biotechnology and Biolegend respectively. HRP conjugated goat anti-rabbit and rabbit anti-rat secondary antibody from Bangalore Genei. Matrigel basement membrane matrix from BD Biosciences, gelatin from amresco and ECL (Super signal Kit) was purchased from PIERCE Biotechnology.