Salmon dna protein a agarose

Cells were grown in 10-cm dishes, then cross-linked with 1% formaldehyde and sonicated. Supernatants were immuno-cleared with salmon DNA/protein A-agarose (Merck Life Science) and immunoprecipitated with anti-Sp1 primary antibody (1C6, Santa Cruz Biotechnology) or nonspecific IgG. Pellets were washed, eluted with a buffer consisting of 1% SDS and 0.1 mol/L NaHCO3, and digested with proteinase K. DNA was obtained by phenol/chloroform extractions and precipitated with ethanol. The yield of target region DNA in each sample after ChIP was analyzed by real-time PCR. The primers used to amplify a region containing a Sp1 site located into the EphA3 promoter sequence were: 5′-CAAACTTGACATCAGCCTGCG-3′ (forward) and 5′-TCTCCATGAAGCATGCCACT-3′ (reverse). Data were normalized to the input for the immunoprecipitation and the results were reported as fold changes with respect to nonspecific IgG. The Sp1 sites within the EphA3 promoter were identified using the program “AliBaba2.1”, which is a specific tool for predicting binding sites of transcription factors in an unknown DNA sequence by constructing matrices on the fly from TRANSFAC 4.0 sites.

Cells grown in 10 cm plates were shifted for 24 h to medium lacking serum, then treated with vehicle and FGF2 for 2 h, and then cross-linked with 1% formaldehyde and sonicated. Supernatants were immuno-cleared with salmon DNA/protein A-agarose (Merck Life Science, Darmstadt, Germany) and immunoprecipitated with anti-c-Rel antibody or nonspecific IgG (Santa Cruz Biotechnology, DBA, Milan, Italy). Pellets were washed, eluted with a buffer comprising 1% SDS and 0.1 mol/L NaHCO₃, and digested with proteinase K. DNA was obtained by phenol/chloroform extractions and precipitated with ethanol. The yield of target region DNA in each sample after ChIP was analyzed by real-time PCR using platform Quant Studio7 Flex Real-Time PCR System (Thermo Fisher Scientific, Monza, Italy). The primers used to amplify a region containing an NF-kB site located into the S100A4 promoter sequence were: 5′-GCAAATGTTCACTGCCCAGA-3′ (Fwd) and 5′-ATCACATCCAGGGCCTTCTC-3′ (Rev). Real-time PCR data were normalized with respect to unprocessed lysates (Input) and the results were reported as fold changes with respect to nonspecific IgG.

Cells were grown in 10-cm dishes, exposed to treatments and then cross-linked with 1% formaldehyde and sonicated. Supernatants were immunocleared with salmon DNA/protein A-agarose (Merck Life Science, Milan, Italy) and immunoprecipitated with anti-p-c-Jun (Thr93) antibody or nonspecific IgG. Pellets were washed, eluted with a buffer consisting of 1%SDS and 0.1 mol/L NaHCO3, and digested with proteinase K. DNA was obtained by phenol/chloroform extractions and precipitated with ethanol. The yield of target region DNA in each sample after ChIP was analyzed by real-time PCR. The primers used to amplify a region containing an AP-1 site located into the p53 promoter sequence were: 5′-GCAGCCATTCTTTTCCTGCT-3′ (Fw) and 5′-CAGTGACCCGGAAGGCAGTC-3′ (Rv), as indicated. Data were normalized to the input for the immunoprecipitation and the results were reported as fold changes respect to nonspecific IgG.