Figure 5.

ChIP assays compare NFκB immunoprecipitates of glomerular DNA from young and old rats. (A) Schematic illustrating primer set sites in relation to the putative NFκB site and the gene transcription start site for VCAM (B), ICAM (C), Pnoc (D), and ceruloplasmin (E). (F) For the nephrin κB site, which was 2.5 kb upstream of the start site, two control primer sets were chosen downstream of the putative κB site as shown in the schematic. NFκB p50 antibodies preferentially coimmunoprecipitated DNA fragments spanning the κB sites of genes from old (24 month) versus young (2-month) glomeruli for five examples of genes expressed in glomerular endothelial cells (VCAM and ICAM), podocytes (prepronociceptin and nephrin), and parietal epithelial cells (ceruloplasmin). Additional controls are shown to demonstrate that DNA sequences not containing κB sites upstream and downstream from the putative κB sites were not preferentially coimmunoprecipitated from old glomerular DNA. Data were analyzed by one-way ANOVA using a Bonferroni correction for multiple comparisons. *P < 0.01; **P < 0.001. The white bars refer to immunoprecipitation with nonimmune rabbit IgG. The closed bars refer to immunoprecipitation using rabbit anti-NFκB p50 IgG.