Time course analysis of LPS-mediated IL-8 gene activation. (A) Total RNA was isolated at indicated time points after LPS administration and used in real-time PCR reactions. Where indicated, HT-29 cells were pre-treated with IFN-γ (10 ng/ml) for 24 hours. The IL-8 mRNA levels were normalized to G6PD levels and expressed as relative to untreated control cells. Data points represent the average of triplicate determinations ± SD. Similar results were obtained in 3 independent experiments. *, p < 0.01; n.s.= not significant, in comparison to control culture without IFN-γ. (B) Lysates were collected at the indicated time points in RIPA buffer and 50 μg of protein samples were loaded for electrophoresis. The expression levels of IκB-α were detected using anti-IκB-α antibodies. The levels of γ-tubulin were used to demonstrate equal loading. Protein levels were quantified using the software Quantity One (Bio-Rad). The IκB-α protein levels were normalized to γ-tubulin levels and expressed as relative to untreated control cells. Data points represent the average of three independent experiments ± SD. A representative blot is shown. *, p < 0.01.
Angrisano et al. BMC Microbiology 2010 10:172 doi:10.1186/1471-2180-10-172