Protein a g plus agarose reagent

Chromatin immunoprecipitation assays were done as described by Svotelis et al. [29 (link)]. 10⁷ shCtrl and shHDAC1 IEC-6 cells were recovered and crosslinked in medium containing 1% formaldehyde for 10 min at room temperature, before stopping the reaction with glycine. Chromatin from lysed cells was sonicated with a BRANSON digital sonifier (model S-250D, Branson Ultrasonics, Danbury, CT, USA), for six cycles of 10 sec (shCtrl cells) and five cycles of 10 sec (shHDAC1 cells) at 15% sonication intensity, to obtain DNA fragments between 300 and 500 bp. Chromatin immunoprecipitation was performed with an antibody against hypo- and hyperphosphorylated forms of RNA polymerase II (CTDH48) (EMD Millipore, Billerica, MA, USA) and with protein A/G PLUS-agarose reagent pre-blocked with BSA (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The input DNA, used to determine the amount of DNA for each immunoprecipitation, represents one percent of the lysate. Immunoprecipitated DNA was diluted 1:10 before semi-quantitative PCR amplification of 70 to 145 bp Ccl2 and Gapdh promoter and gene exon 2 downstream sequences (Additional file 2), for 30 or 32 cycles with the touchdown amplification protocol used for chemokine expression analysis. Results are representative of three independent experiments.

Protein–protein interactions were evaluated by co-immunoprecipitation analysis as previously described [8 (link), 28 (link), 35 (link), 36 (link), 42 (link)] with minor modification. Briefly, cell extracts were incubated overnight with corresponding antibodies and then incubated for 3 h with 20 µl of the Protein A/G Plus-Agarose reagent (Santa Cruz Biotechnology). Immunocomplexes were washed four times in buffer containing 50 mM Tris–HCl (pH 7.6), 150 mM NaCl and 0.1% Triton X-100. The immunoprecipitates were separated by SDS-PAGE followed by transfer to nitrocellulose membranes. The membranes of immunoprecipitates were probed with use of corresponding antibodies.