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. 2009 Oct 30;9(5):192–202. doi: 10.4110/in.2009.9.5.192

Figure 4.

Figure 4

Induced iNOS expression is dependent on TLR2 but not TLR4. (A) MDMs were pre-incubated with anti-TLR2, anti-TLR4, or isotype-control mAbs (10 µg/ml), followed by treatment with IFN-γ, 1,25-D3, or both for 18 h. Cells were then infected with MTB H37Ra (MOI=1) for 18 h. Western blot analysis was performed using rabbit anti-iNOS Ab. (B) Effects of specific hTLR2 or hTLR4 gene silencing on MTB H37Ra -stimulated iNOS expression of human MDMs pretreated with IFN-γ plus 1,25-D3. Gene silencing using shRNAs generated by psiRNA-h7SKGFPzeo plasmids was performed by transient transfection using amaxa Nucleofector technology. At 18 h after transfection, the cells were treated with IFN-γ plus 1,25-D3 for 18 h, followed by stimulation with MTB H37Ra (MOI=1) for 30 min. Cell lysates were prepared and analyzed on Western blots for the expression of iNOS, TLR2, TLR4, IkB-α, and p-IKKα/IKKβ. The β-actin level was used as a control. Results are representative of three experiments.