Anti rela antibody

ChIP and Re-ChIP assays were performed using the ChIP-IT^® Express Chromatin Immunoprecipitation Kits and Re-ChIP-IT^® kits (Active Motif), according to the manufacturer’s instructions. Chromatin samples were immunoprecipitated with either anti-RelA antibody (Cell Signaling Technology) or normal mouse IgG (Cell Signaling Technology) as a negative control. Precipitated DNA was amplified by PCR using primers provided in the Additional file 1: Table S1. Non-immunoprecipitated chromatin fragments were used as an input control.

HeLa cells synchronized at G1/S or S-phase were washed with room temperature PBS and lysed with modified RIPA buffer (50 mMTris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40) including a 1:100 dilution of Protease Inhibitor cocktail (Sigma, UK), PMSF and phosphatase inhibitor (Phos Stop, Roche). Immunoprecipitation was carried out using Immunoprecipitation kit-Dynabeads Protein G (Invitrogen) with anti-RelA antibody (#3034, Cell Signaling, MA, USA). The samples were analyzed by western blotting using anti-E2F-1(Cell Signaling, #3742) or anti E2F-4 (Santa Cruz, C-20 sc-866) antibodies.

The ChIP assay was carried out following the manufacturer’s protocol (SimpleChIP Enzymatic Chromatin IP Kit, Cell Signaling, 9002). Briefly, primary corneal epithelial cells were crosslinked with 1% formaldehyde and blocked with glycine, washed, and digested by micrococcal nuclease. The nuclear pellet was suspended in ChIP buffer and sheared by sonication. An aliquot of sheared chromatin sample was set aside as input control. The remained chromatin was then incubated with anti-RelA antibody (Cell Signaling, 8242). Normal Rabbit IgG (Cell Signaling, 2729) was used as a negative control. The immunoprecipitated chromatins were then eluted with ChIP elution buffer. The DNA fragments were released by treatment with proteinase K at 65°C. The released DNA fragments were purified with columns and amplified by site-specific primers by quantitative PCR assay. The data were analyzed by the following formula: percent (%) input recovery = (100/(input fold dilution/bound fold dilution)) × 2(input CT - bound CT). Pairs of primers designed to amplify the specific target sequences of the putative promoters are listed in Table 2.