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. 2016 Apr 7;44(12):5658–5672. doi: 10.1093/nar/gkw189

Figure 5.

Figure 5.

Butyrate differentially regulates Sp1 and Sp3 binding to TLR5 promoter. (A) Immunoblots showing Sp1 and Sp3 expression in the cytosolic and nuclear extracts from the untreated- and butyrate-treated (4 mM, 24 h) HT29 cells. Histone H3 and Tubulin were probed as loading controls for the nuclear and cytosolic extracts, respectively. (B and C) EMSA showing retarded bands due to binding of Sp1 and Sp3 present in the nuclear extracts of HT29 cells to the biotin-labeled oligonucleotides containing the SP-A and/or SP-B sequences. Probes used contained both SP-A and SP-B sequences (B). Mutated or 100-fold excess of the unlabeled oligo was used as the competitor. For supershift (ss), reaction was carried out in presence of antibodies (2 μg) to Sp1, Sp3 or both. (D) DAPA. Biotin-labeled TLR5 minimal promoter was incubated with the nuclear extracts of untreated or butyrate-treated (4 mM for indicated times) HT29 cells. DNA-protein complexes were pulled down with streptavidin-coated beads, separated by Western blot and probed with Sp1 and Sp3 antibodies. Total proteins in the nuclear extracts were immunoblotted as loading controls. (E and F) ChIP assays with IgG, Sp1 and Sp3 antibodies in butyrate-treated (4 mM for indicated times) or untreated HT29 cells. qPCR showed IgG, Sp1 and Sp3 binding to endogenous (E) TLR5 promoter or (F) GAPDH promoter. *P < 0.05; **P < 0.01 compared with untreated.