Sc 11350x

Chromatin immunoprecipitation (ChIP) assays were carried out with the ChIP-IT Kit (Active Motif, Carlsbad, CA) using ∼1.5 × 10⁷ ATDC5 murine chondrocytes. The ATDC5 were cultured using different conditions, which included: 1) hypertrophic differentiation for 6 days using a 1:1 mixture of DMEM and Ham’s F12 medium supplemented with 5% FBS, 1% Anti-Anti, 50 µg/mL ascorbic acid, and 0.5 mmol/L NaH₂PO₄, 2) cells at 70–80% treated with CML-BSA (200 μg/mL) for 3 days or unmodified BSA (200 μg/mL) for 3 days, and 3) cells grown in HG (25 mmol/L) media for 5 days or mannitol (25 mmol/L) as osmotic control. Cells were fixed with formaldehyde and nuclei obtained following Dounce homogenization. ChIP was performed following the manufacturer’s instructions using an anti-FOXO1 antibody (5 μg) (SC-11350X; Santa Cruz Biotechnology) or control polyclonal nonspecific IgG (Cell Signaling Technology). Chromatin–antibody complexes were purified using protein G–coupled beads. Three quantitative real-time PCR reactions for the RANKL gene promoter with primers used were: forward, 5′-TGAAGACACACTACCTGACTCCTG-3′ and reverse, 5′-CCCACAATGTGTTGCAGTTC-3′, which flanks a consensus FOXO1 response element. The experiment was repeated three times with similar results.