Magna chip a g one day chromatin immunoprecipitation kits

MyoD and MEF2D ChIP was performed with Magna ChIP™ A/G One-Day Chromatin Immunoprecipitation Kits (Millipore, Bedford, MA) according to the manufacturer’s instructions. Briefly, the ChIPed DNA was eluted, reverse X-linked, purified, and analyzed by qRT-PCR. Data represent the mean ± SEM of three independent experiments.

MC3T3-E1 cells were transduced with the pINDUCER20-Prdm16 virus and selected with G-418 to generate cells with inducible expression of Prdm16. Cells were maintained in the medium without doxycycline. Cells were seeded at 5 × 10⁴ cells per cm² into 20 mm Petri dishes with or without 1 μM doxycycline. Twenty-four hours later, the medium was changed to a serum-free medium with or without 1 μM doxycycline, and 24 h later the medium was changed to a serum-free medium with or without 1 μM doxycycline and 10 ng μl⁻¹ TGF-β1 (R&D Systems). Finally, 24 h later, the cells were collected with Magna ChIP A/G One-Day Chromatin Immunoprecipitation Kits (Millipore). The sheared chromatin was immunoprecipitated with 10 μg ml⁻¹ anti-Prdm16 antibody (R&D Sysytems, AF6295), 10 μg ml⁻¹ anti-Smad3 antibody (Abcam, ab28379), 10 μg ml⁻¹ anti-Acetyl-Histone H3 antibody (Millipore, 17–615) or 10 μg ml⁻¹ anti-IgG antibody (Millipore, PP64-1KC). DNA was quantified by qRT–PCR with different primer sets and normalized with input DNA of each group. The 2 kb upstream region from the mouse Sost transcription start site was divided into six regions (S1–S6). Only the S3 region (Fig. 3f,1,036–1,328 bp upstream from transcription start site) exhibited significantly difference among treatments.