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. 2015 Mar 6;19(5):1065–1076. doi: 10.1111/jcmm.12500

Figure 5.

Figure 5

Interference of Gal-9 expression impaired DV-induced activation of NF-κB. Human DCs were transfected with control siRNA (si-Ctl) or Gal-9 siRNA (si-G9-1) for 24 hrs and then infected by mock or DV for an additional 24 hrs. The nuclear extracts were collected for determination of expression of c-Rel, t-p65 (total p65), t-p50 and USF-2 (an internal control) by Western blotting (A). To exclude the possibility of contamination of membrane proteins, the samples were also probed with antibodies against Na+/K+-ATPase α, a membrane marker. The positive control was the cellular extract, after removing cytoplasmic portion, treated with RIPA buffer in the presence of Triton-X100. Such a preparation contained membrane proteins (A). The densitometry data pooled from three independent experiments using different donor cells were analysed (B). The DNA-binding activity of NF-κB in nuclear extracts was determined by EMSA (C). Densitometry data pooled from three independent experiments using different donor cells were analysed (D). Nuclear extracts pre-incubated with wild-type or mutant κb oligonucleotides served as controls. In (E), similar to those in (A), the levels of both nuclear p52 and RelB were examined. To control the equal amounts of loaded nuclear proteins, the levels of USF-2 protein were used as the backgrounds to normalize individual band intensities shown in EMSA (data not shown). Cpt, competitors; Wt, wild-type; Mt, mutant. The analysis was performed by anova. *P < 0.05, **P < 0.01. Ctl, control.