Figure 4.

TNF α does not alter Sp3 binding to the AQP3 promoter in HT‐29 cells. Chromatin immunoprecipitation (ChIP) was performed on HT‐29 cells treated with TNF α (25 ng/mL) for 4 h. ChIP experimental success was verified using IP of Histone H3 bound to the Ribosomal protein L30 (RPL30, 160 bp) promoter in untreated samples (A). Sp3 antibody was used to IP chromatin, with an equivalent amount of rabbit IgG isotype antibody used as a negative control for nonspecific IP, and PCR was used to amplify the AQP3 promoter (AQP3, 101 bp) from the precipitate (B). Two percent of the total input into the ChIP reaction was used as a loading control to assess the equivalence of sample input, whereas a no template control (NTC) was used to ensure specific signal amplification in the PCR reaction. Relevant molecular weight markers from a 1 kb ladder (L) are shown on the left of the gels. A representative image for the ChIP results obtained is shown (n = 3).