Magna chip a g kit

Immunoprecipitation was performed using a specific Bmal1 antibody (Abcam AB93806)^{15 (link)} or control rabbit IgG with Magnetic Protein A/G beads (Magna ChIP A/G kit, Millipore), as described previously.^{15 (link),26 (link)} Differentiated C2C12 myotubes chromatin was sonicated and purified following formaldehyde fixation. Real-time PCR was carried out in triplicate using purified chromatin with specific primers for predicted Bmal1 binding E or E’-box elements identified within the gene regulatory regions. Primers flanking known Bmal1 E-box in Rev-erbα promoter were used as a positive control and TBP first exon primers as a negative control. Chromatin immunoprecipitation (ChIP) primer sequences are shown in Supporting Table S2. Fold enrichment over IgG was expressed normalized to 1% of input.

Chromatins were prepared from AC or cultured articular chondrocytes and cross-linked with 1% formaldehyde. ChIP assays were performed according to the manual of Magna ChIP A/G kit (Millipore) using a ChIP grade NFAT1 antibody (Santa Cruz, Sc-13034). To identify potential NFAT1-binding target genes, specific primers were designed from Acan, Col2a1, Col9a1, Col11a1, Ilb1, Mmp13, and Tnfa promoter sequences encompassing NFAT1 binding sites (Table 2). Histone methylation ChIP assays were performed using chromatins prepared from articular chondrocytes with dimethylated histone 3 lysine 4 (H3K4me2, Millipore) or H3K9me2 (Millipore) antibodies. Normal mouse IgG was used as a baseline control to confirm the specificity of the ChIP assays. Chromatin prepared from femoral head AC of Nfat1^−/− mice was used as a negative control for the NFAT1 ChIP assay. Input, immunoprecipitated and control samples were analyzed by qPCR using specific primers designed around the transcription start site (TSS) of the Nfat1 gene (Table 2). The relative enrichment levels of ChIPed samples were first normalized to input samples and were then compared to the relative enrichment levels of IgG control samples.

Chromatin immunoprecipitation (ChIP) assays were performed with the Magna ChIP A/G kit (Millipore, Temecula, CA, USA). Cells were fixed in 1% formaldehyde for 10 minutes and DNA fragmented by means of sonication (five 10-second pulses). Samples were diluted in ChIP dilution buffer and incubated with 4μg of Brd2 or Brd4 antibody (Bethyl Laboratories) and protein A/G magnetic beads overnight. Antibody/DNA complexes were captured, washed, eluted, and reverse cross-linked. DNA was purified by means of spin columns containing activated silica membrane filters. The precipitated DNA was re-suspended, and real-time PCR was performed using primers spanning ARE sites on the HO-1 and NQO1 gene promoters. The primers used were: NQO1 F: 5′-CAGTGGCATGCACCCAGGGAA-3′, R: 5′-GCATGCCCCTTTTAGCCTTGGCA-3′ (28 (link)) and HO-1 F: 5′-CTGCCCAAACCACTTCTGTT-3′, R: 5′-ATAAGAAGGCCTCGGTGGAT-3′ (29 (link)). DNA expression was normalized to input DNA for each sample.