Western blotting detection reagents

The levels of cyclinD1, cdk4 Nox1, Nox2, Nox4, p47^phox, c-Src and growth factor receptors were determined by Western blotting using specific antibodies as described in detail earlier [19 (link), 24 (link)]. Equal amounts of protein (30 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes and incubated with the respective primary antibodies: cyclin D1, Cdk4, c-Src, phospho-c-Src, EGF-R, phospho-EGFR, PDGF-R β, phospho-PDGF-R β, IGF-R β, phospho-IGF-IR, Nox1, Nox2, Nox4 and phosphor-p47^phox. The antibody-antigen complexes were detected by second antibody horseradish peroxidase-conjugated goat anti-mouse, donkey anti-goat and goat anti-mouse. Protein bands were visualized by enhanced-chemiluminescence(ECL). Western blotting detection reagents were from Santa Cruz Biotechnology. Quantitative analysis of specific bands was performed by densitometric scanning of the autoradiographs with an enhanced laser densitometer (LKB Ultroscan XL, Pharmacia, Dorval, Qc, Canada) and quantified by using gel-scan XL evaluation software (version 2.1) from Pharmacia.

Proteins separated by SDS-PAGE were transferred onto a PVDF membrane which was then blocked (5% non-fat dried milk and 0.1% Tween-20 in PBS) for 1 hr at room temperature. The membrane was incubated with primary antibody diluted in PBST with 2% bovine serum albumin, followed by washing with PBST for 5 min trice, incubated with goat anti-rabbit-horseradish peroxidase conjugated secondary antibody (1:20000; Santa Cruz Biotechnologies), or rabbit anti-mouse- horseradish peroxidase conjugated secondary antibody (1:5000; Santa Cruz Biotechnologies) diluted in PBST for 1 hr at room temperature, and then visualized with western blotting detection reagents (Thermo Fisher Scientific).