Figure 6.

In vivo binding of NFκB (RelA) to its cognate motifs on human hnRNPD promoter. (A) Schematic representation depicting the positions of RelA (NFκB) binding motifs − 1358/− 1347 in the hnRNPD promoter and primers used for ChIP assay. Cross linked chromatin from PDTC treated or untreated SCC-4 cells was immunoprecipitated with anti-RelA antibody and subjected PCR after reversal of cross linking and sonication using the primers depicted in the supplementary Fig. S3. Sonicated genomic DNA from SCC-4 cells was used as input. Chromatin immunoprecipitated with Anti-pol II antibody was subjected to PCR using GAPDH primers and served as positive control. While PCR with gene specific primers and chromatin immunoprecipitated using normal mouse IgG served as negative control (IgG lane). Similarly, PCR performed with gene specific primers without a template also served as control. A 50 bp DNA ladder was used to determine the size of PCR fragments on agarose gel. (B) Representative agarose gel image of ChIP assay for RelA (NFκB) binding motifs − 1358/− 1347. (C) Densitometric quantification PCR fragment of ChIP assay for RelA (NFκB) binding motifs − 1358/− 1347. Values are mean ± SD from there independent experiments. Results were analyzed by Student’s t test and values significantly different from respective controls have been marked by stars (*P ≤ 0.05, ****P ≤ 0.0001). (D) Schematic representation depicting the position of RelA (NFκB) binding motif (− 1052/− 1041) in hnRNPD promoter) and primers used for ChIP assay. (E) Representative agarose gel image of ChIP assay for RelA (NFκB) binding motifs (− 1052/− 1041). (F) Densitometric quantification PCR fragment of ChIP assay for RelA (NFκB) binding motifs (− 1052/− 1041). Values are mean ± SD from three independent experiments. Values significantly different from respective controls have been marked by stars (**P ≤ 0.01, ***P ≤ 0.001). Other details are same as described for RelA (NFκB) binding motifs − 1358/− 1347.