Anti smad1 5 8

Anti-p-PDHE1α (S293, S300) (Calbiochem, San Diego, CA, USA, AP1062-1064, respectively) were used for immunohistochemistry. Anti-p-PDHE1α (S293, S300) (Abfrontier), Anti-SMAD1/5/8 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-6031-R), anti-p-SMAD 1/5/8 (Cell Signaling Technology, Danvers, MA, USA, #9511), COXIV (Abcam, Cambridge, UK, ab16056) and anti-Flag (Sigma-Aldrich, F1804) antibodies were used for both immunofluorescence and western blotting. Anti-SMAD1 (Invitrogen, Carlsbad, CA, USA, #38-5400), anti-SMAD5 (Abgent, San Diego, CA, USA, AJ1726a), and Anti-SMAD1/5 (Santa Cruz Biotechnology, sc-6201) were used for immunofluorescence. Anti-α-tubulin (Applied Biological Materials, G098), anti-lamin B (Santa Cruz Biotechnology, sc-6216), anti-SMAD4 (Cell Signaling Technology, #9515) and anti-PDK4 serum (Abfrontier) were used for western blotting.

C3H10T1/2 cells were obtained from ATCC (American Type Culture Collection, Manassas, VA). Recombinant adenovirus expressing exogenous BMP9 (Ad-BMP9) was kindly provided by Dr. Tong-chuan He of the University of Chicago Medical Center. The NICD overexpression plasmid was purchased from Genechem (Genechem Co., LTD, Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were both obtained from Gibco (Grand Island, NY, USA). DAPT was purchased from Cell Signaling Technology (CST; Danvers, MA, USA), and TPA was obtained from Beyotime (Beyotime Biotechnology, Shanghai, China). Anti-OCN, anti-JunB, anti-Runx2, anti-OPN and anti-β-actin antibodies were purchased from Abcam (MA, USA). Anti-Smad1/5/8, anti-p-Smad1/5/8, anti-PCNA, and anti-Cyclin D1 antibodies were obtained from Santa Cruz Biotechnology, Inc. (CA, USA). An anti-NICD antibody was purchased from CST. Unless otherwise indicated, all chemicals were purchased from Sigma-Aldrich (Saint Louis, USA).

Cells and tissues were gently lysed with lysis buffer for 1 h on ice and then centrifuged at 15,000 g and 4 °C for 15 min, and the supernatant was collected. The soluble lysates were incubated with mouse anti-Jab1 antibody (#sc-13157, Santa Cruz Biotechnology) at 4 °C, and then with Protein A/G bead (Santa Cruz Biotechnology) and washed times. Immune complexes were eluted by boiling for 10 min at 95 °C in SDS sample buffer, followed by Western blot analysis with anti-Jab1 (1:1000, Cell Signaling), anti-Runx2 (1:1000; Cell Signaling Technology), or anti-Smad1/5/8 (1:1000; Santa Cruz Biotechnology) antibodies.