P smad 1 5 8 antibody

Cellular fractionation was performed using NE-PER Nuclear and Cytoplasmic Extraction reagents (Pierce Biotechnology, Inc., Rockford, IL, USA), according to the manufacturer's instructions. Subsequently, fractions were processed, as described above for western blotting. The membranes were incubated with either rabbit anti-Runt-related transcription factor (Runx)-2 polyclonal antibody (1:500; Santa Cruz Biotechnology, Inc.; cat. no. sc-10758), or rabbit anti-p-small mothers against decapentaplegicp (p-Smad) 1/5/8 antibody (1:1,000; Cell Signaling Technology, Inc.; cat. no. 9516) overnight at 4°C. The subsequent steps were performed as described above. To normalize the bands, the filters were removed and re-probed with anti-paxillin (1:1,000; BioLegend, Inc., San Diego, CA, USA) and anti-B23/nucleophosmin (Santa Cruz Biotechnology, Inc.) antibodies. The density of the bands were quantified densitometrically. Densitometric quantification of the protein bands was analyzed by TINA software version 2.1 (Raytest Isotopenmessgeräte GmbH, Straubenhardt, Germany).

For either fish embryo or cultured cells, total protein was extracted using an extraction buffer (63mM Tris-HCl, PH6.8, 10% glycerol, 5% β-Mercaptoethanol, 3.5% SDS) containing 1X Complete (Roche, 11873580001). Western blotting was performed as described previously [27 (link)] using corresponding antibodies as indicated in the figures. Actin antibody (Huabio, R1207-1), GAPDH antibody (Huabio, M1211-1), p-Erk antibody (Cell Signalling Technology, 9101), total Erk antibody (Cell Signalling Technology, 4695), pSmad1/5/8 antibody (Cell Signalling Technology, 9511), Bip antibody (Sigma, G9043), Chop antibody (Sigma, G6916), phosphorylated eIF2a (p-eIF2a) antibody (Cell Signaling Technology, 9721S), and total eIF2a antibody (Cell Signaling Technology, 9720S), and Flag antibody (Sigma, F1804) were purchased from the companies as indicated. Signal intensity of a desired band was calculated by ImageJ software (v.1.48).