Phospho smad1 5 8

Culture materials and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). MAPK inhibitors including PD98059 (inhibitor for ERK), SP600125 (inhibitor for JNK), SB203580 (inhibitor for p38), and LDN193189 (inhibitor for ALK3) were purchased from Calbiochem (La Jolla, CA, USA). Antibodies against p38, phospho-p38, SMAD1/5/8, phospho-SMAD1/5/8, osterix, and OPN were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibody against Runx2 was purchased from Cell Signaling Technology (Beverly, MA, USA). The catalog numbers and dilutions for antibodies used in Western blot analysis were provided in Table S1. Neutralizing mABs against BMP-2, BMP-6 were purchased from R&D Systems (Minneapolis, MN, USA). Runx2, SMAD1 siRNA vectors, and a control siRNA construct (containing random DNA sequences) were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals of reagent grade were obtained from Sigma (St. Louis, MO, USA).

Whole cell extracts were prepared using radioimmunoprecipitation assay buffer (RIPA; Thermo Scientific, Rockford, IL) containing a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Protein quantification was done using a BCA protein assay kit (Pierce) according to the manufacturer's instructions. Total cell protein (15-20 μg) was used for western blotting. Samples were transferred to PVDF membranes, then immersed in 3% skim milk with antibodies against BMP-2 (#ab14933, Abcam, Cambridge, MA), Smad1/5/8 (#12656 Cell Signaling Technology), phospho-Smad1/5/8 (#sc-12353-R, Santa Cruz), p21^WAF1/CIP1 (#2947, Cell Signaling Technology), p27^KIP1 (#2552, Cell Signaling Technology), Cdk2 (#2546, Cell Signaling Technology), Caspase-3 (#9662, Cell Signaling Technology), Cleaved Caspase-3 (#9661, Cell Signaling Technology), and GaDD45α (#4632, Cell Signaling Technology) overnight at 4°C. Blots were washed in TBS containing 0.1% Tween20 and labeled with horseradish peroxidase conjugated secondary anti-rabbit antibody (Cell Signaling Technology). Specific complexes were visualized with an ECHO chemiluminescence (ECL) detection system (GE Healthcare, Little Chalfont, UK) using the Chemidoc imaging system (Bio Rad, CA, USA). The protein expression levels are expressed relative to GAPDH.