Figure 6.

Effects of RPS3 Gene Silencing on NF-κB Translocation and Activity

(A) Nuclear translocation of p65 induced by CSE was captured using immunofluorescence staining in RAW 264.7 cells. Percentage of cells with p65 nuclear staining was quantified (n = 4 separate experiments). (B) Immunoblot of nuclear NF-κB subunit p65 and RPS3 accumulation. Mouse lung nuclear proteins were separated by 10% SDS-PAGE, and probed with anti-p65, anti-RPS3, or anti-TATA binding protein (TBP) mAbS. TBP was used as a nuclear protein loading control (n = 9 mice per treatment group). SA, sham air exposed. CS, CS exposed. (C) Nuclear p65 DNA-binding activity was determined using a TransAM p65 transcription factor ELISA kit (n = 9 mice per treatment group). (D) NF-κB reporter gene assay in NF-κB/SEAP reporter RAW 264.7 cells pre-treated with RPS3 siRNA and then stimulated with CSE. Results are expressed as fold change relative to media control. The SEAP assay was conducted in duplicate with three independent experiments. Values are shown as means of triplicate ± SEMs. *Significant difference from control siRNA, p < 0.05; ^#significant difference from the SA or control media group, p < 0.05.