841346.fig.003a
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841346.fig.003b
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841346.fig.003c
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Figure 3: Analysis of IFN-β gene regulation in Mtb-infected or LPS-treated human monocytes. (a) Total cell extracts were prepared at different times following infection with Mtb or LPS treatment. Whole cell extracts (30 μg) were analyzed on an SDS-7% PAGE gel and subjected to immunoblot analysis with anti-IRF-3 antibody to detect the phosphorylated IRF-3 isoform (IRF-3P; upper panel). The total content of IRF-3 was evaluated as an internal loading control (IRF-3C; lower panel). The results shown are from one of three experiments performed with cell extracts from different monocyte cultures that yielded similar results. (b) Nuclear extracts were prepared from Mtb-infected at different time points or from cells treated for 1 hr with LPS. Five μg of nuclear proteins were subjected to EMSA analysis using as oligonucleotide the κB-IFN-β sequence. Supershift assays were performed after incubation with anti-p65 Abs as indicated (ss). (c) RNA was extracted from monocytes infected with Mtb (MOI = 1) or treated with LPS (1 μg/ml) for 8 hr. Real-time reverse transcription-polymerase chain reaction was performed to measure the expression of IFN-β. The results are shown as a ratio to the GAPDH level and represent the mean ± SE of triplicate values. The results shown are from one out of three experiments performed with different donors that yielded similar results.