Sc 293138

Metatarsals were isolated from 8 newborn C57BL/6J mice and cultured in DMEM (Gibco) containing 1% heat-inactivated fetal calf serum (Invitrogen) and 100 U/ml penicillin-strep tomycin in the presence of BMP2 (300 ng/ml) and siPERK, siATF4, or siPERK + siATF4 for 5 days, followed by histological examination. Affinity-purified anti-caspase-3 (ab32351; Abcam), caspase-12 (ab62484; Abcam), anti-CHOP (ab11419; Abcam) and anti-p-JNK (sc-293138; Santa Cruz Biotechnology, Inc.) were diluted at 1:100 and sections were incubated at room temperature for 2 h. For detection, biotinylated secondary antibody (sc-2364) and HRP-streptavidin complex (sc-2363) (Santa Cruz Biotechnology, Inc.) were used. A total of 0.5 mg/ml 3,3′-diami-nobenzidine (DAB) in 50 mM Tris-HCl substrate (Sigma) was used for visualization, and the sections were counterstained with Mayer's hematoxylin (H9627; Sigma).

Proteins in total cell extract from micromass cultures of BMP2-treated (300 ng/ml) ATDC5 or C3H10T1/2 cells were resolved on a 10% SDS-polyacrylamide gel and electroblotted onto nitrocellulose membranes. After blocking in 10% non-fat dry milk in Tris-buffered saline Tween-20 [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% Tween-20], the blots were incubated with either mouse monoclonal anti-PERK antibody (sc-377400; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-C/EBP homologous protein (CHOP) antibody (ab11419; diluted 1:1,000; Abcam, Cambridge, UK) or rabbit anti-caspase-3 antibody (ab32351; Abcam) and anti-p-c-Jun N-terminal kinase (JNK) antibody (sc-293138; Santa Cruz Biotechnology, Inc.) for 1 h. After washing, the respective secondary antibody [horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin or HRP-conjugated anti-rabbit immunoglobulin; Sigma. St. Louis, MO, USA; A9044] was added, and the bound antibody was visualized using an enhanced chemiluminescence system (Amersham Biosciences, Uppsala, Sweden).