Figure 4. Analysis of promoter binding activity by NFκB subunits using ChIP assay. ChIP was performed with 200 μg lysate. A: Binding of NFκB p65 and p50 was observed at both 1 and 4 h post infection on the IL-8 promoter (left and right panels, respectively). A negligible amount of cREL bound to the IL-8 promoter at 1 and 4 h. Inhibitor pretreatment reduced the binding of p65, p50, and cREL on the IL-8 promoter at both time points post infection. For ChIP, 5% of the total lysate was used as a loading control (Load) to show that an equivalent amount of extract was subjected to immunoprecipitation. Isotype control is shown (Ctrl). B: p65 binding to the MCP-1 promoter was not observed at 1 or 4 h post infection. p50 bound but showed no apparent change across the pretreatment groups at 1 or 4 h. Binding of cREL on the MCP-1 promoter was apparent at 1 h but increased dramatically at 4 h post infection.