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. 2019 Jun 2;15(8):1571–1581. doi: 10.7150/ijbs.34211

Figure 4.

Figure 4

Caffeine inhibits NF-κB activation in THP-1 macrophages stimulated by LPS. (A) The protein levels of IκB-α, p65 and their phosphorylated forms (P-IκB-α and P-p65) were analyzed by western blotting in THP-1 macrophages pretreated with caffeine for 1 h and then incubated with LPS (1 μg/ml) for 3 h. (B) Densitometric analysis was used to quantify the phosphorylation of IκB-α at Ser32. (C) Densitometric analysis was used to quantify the phosphorylation of p65 at Ser536. The results represent the mean ± SD for three experiments. *** p< 0.001 vs. the control group. # p<0.05, ## p<0.01, and ### p< 0.001 vs. the LPS group. (D) p65 nuclear localization was visualized by immunofluorescence analysis with an anti-p65 (green) antibody in THP-1 macrophages pretreated with caffeine (800 μM) for 1 h followed by LPS (1 μg/ml) for 3 h. DAPI (blue) was used as a nuclear marker. Scale bar: 50 μm.