Anti smad1 antibody

Western blot analysis were carried out by using the following primary antibodies: rabbit polyclonal anti-phosphorylated Smad1,5,8 (1:1000, Cell Signaling Technology, code: 9511; lot number:14) rabbit polyclonal anti-Smad1 antibody (1:1000, Cell Signaling Technology, code: 9743; lot number: 2) rabbit polyclonal anti-VEGF antibody (1:1000, Abcam, code: ab46154; lot number: GR218557-1), mouse monoclonal anti-β-actin antibody (1:50000, Sigma Aldrich, code: A5441; lot number: 061M4808), rabbit polyclonal anti-Phospho-VEGF receptor 2 (P-VEGFR2) antibody (1:1000, Cell Signaling Technology, code: 2478; lot number: 13) or rabbit polyclonal VEGFR2 (1:1000, Cell Signaling Technology, code: 2479; lot number: 18). After incubation with primary antibodies, filters were washed and incubated with secondary peroxidase-coupled antibodies anti-rabbit (1:6000, Calbiochem, Milan, Italy, code: 401393; lot number: D00051552) or anti-mouse (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA, code: sc-2005; lot number: H0513). Immunostaining was revealed by enhanced chemiluminescence luminosity (GE Healthcare, Milan, Italy).

C2C12 cells were plated onto 35 mm culture dishes at a concentration of 1 × 10⁵ cells/dish. On the next day, cells were treated with 75 ng/mL of rhBMP-2 combined with 4 μg/mL of LRR2-3 synthetic peptide. At 5, 15, 30, 120, 240 and 480 min, cells were washed with RIPA buffer and centrifuged. Twenty microliters of the supernatant was mixed with 3 × SDS sample buffer, applied to 4–12% SDS-PAGE followed by Western blot analyses with anti-phospho-Smad1/5/9 antibody (Cell Signaling Technology, MA), or anti-Smad1 antibody (Cell Signaling Technology, MA). Signals were detected by immunoreactivity and were analyzed by ImageQuant^TM LAS 4000 (GE Healthcare, IL). The levels of Smad1/5/9 phosphorylation was calculated relative to total Smad1 level of each sample.