Rabbit anti actin polyclonal antibody

Total expression levels of protein/inositol phosphatases including Lyn, Dok1, Ship1, and Shc1 (Santa Cruz biotechnology) were measured upon pulsed stimulation. Lysis buffer supplemented with phosphatase inhibitor and protease inhibitor (1:100) was used to lyse cells. Protein concentrations were measured using Bradford assay (Bio-Rad) and samples were analyzed by Western blot as previously described^{38 (link)} with primary antibodies from Santa Cruz biotechnology. For detection of Shc1 knockdown by western blot, rabbit anti-Shc1 primary antibody from BD Biosciences and Pierce HRP-conjugated polyclonal goat anti-rabbit secondary antibody were used. All blots were re-probed with rabbit anti-actin polyclonal antibody (Novus) and visualized with an Alpha Innotech (San Leandro, CA) imager using a Super- Signal West Femto sensitivity substrate (Thermo Scientific). The relative intensity (RI) was calculated as the quotient of the densitometry signal for the target protein band divided by that for actin, then normalized by the ratio obtained for the control sample.

Samples were lysed and analyzed by Western blotting as previously described (11 (link)). Primary antibody for β-catenin knockdown experiments was rabbit polyclonal anti-β-catenin (H-102; Santa Cruz Biotechnology). Primary antibody to measure levels of β-catenin activation was non-phospho (active) β-catenin (Ser33/37/Thr41) (D13A1) rabbit monoclonal antibody (Cell Signaling). All blots were reprobed with rabbit antiactin polyclonal antibody (Novus). The relative reduction index (RI) was calculated as the quotient of the densitometry signal for the target protein band divided by that for actin, which was then normalized by the ratio obtained with scrambled siRNA or no-ligand controls (considered to be 1).