Western Blotting of Signaling Proteins

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.