Westernbright sirius chemiluminescent hrp substrate

Cells were lysed in RIPA buffer containing protease inhibitor cocktail (Roche) and 1 mM phenylmethylsulfonyl fluoride (PMSF). Equal amount of protein lysate was resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Millipore) using fast Turbo Transfer (Bio-rad). After blocking in 5% skimmed milk for 1 h, the appropriate primary antibody was added: anti-ADAR2 (RED1) (1:1000, ab64830, Abcam), anti-SRp30c(SRSF9) (1:1000, sc-134036, Santa Cruz), anti-FLAG (1:2000, Clone M2, Sigma), anti-cMyc (1:1000, sc-789, Santa Cruz), anti-V5 (1:2000, ab9116, Abcam) or anti-β-actin (1:1000, clone C4, sc-47778, Santa Cruz). Primary antibodies were incubated overnight in the cold room. After washing with phosphate-buffered saline (PBS)/0.2% Tween-20 (PBST), a secondary antibody, namely horse-radish peroxidase (HRP)-conjugated anti-rabbit IgG (1:5000, NA934V, Amersham) or HRP-conjugated anti-mouse IgG (1:5000, NA931V, Amersham), was added for 1 h. After washing with PBST, signals were detected using the WesternBright Sirius Chemiluminescent HRP Substrate (Advansta).