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. 2013 Feb 28;8(2):e57795. doi: 10.1371/journal.pone.0057795

Figure 5. Effect of RSUME195 and RSUME267 over NF-κB signaling pathway.

Figure 5

A. COS-7 cells were co-transfected with RSUME and IκBα, inmunoprecipitated with anti-Flag antibody, and subjected to western blot with anti-V5 or anti-Flag antibodies. *, band corresponding to an IgG used in the inmunoprecipitation. B. and C. COS-7 cells were co-transfected with 500 ng of NF-κB-LUC reporter vector (B), or IL-8-LUC or IL-8(NF-κBmut)-LUC reporter vector (C), 300 ng of Gaussia as control and different concentrations of RSUME195 or 267 vectors (100 or 500 ng). After 24 h cells were stimulated with 10 ng/ml TNF-α for 6 h and luciferase (LUC) activity was measured in the cell extracts. Each value was normalized to Gaussia value. Results are expressed as mean ± SEM from triplicates of one representative experiment of three experiments with similar results. *, p<0.05 compared with cells transfected with the empty vector (Vect) stimulated with TNF-α (ANOVA with Scheffè’s test). #, p<0.05 compared each concentration of cells, stimulated with TNF-α, transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test). D. IL-8 mRNA level was analyzed by quantitative real-time RT-PCR in triplicates in HepG2 cell stimulated with 10 ng/ml TNF-α for 6 h, and the values are given as mean ± SEM after normalization to RPL19. *, p<0.05 compared with cells transfected with the empty vector (Vect) (ANOVA with Scheffè’s test). #, p<0.05 compared the condition transfected with RSUME195 vs. RSUME267 (ANOVA with Scheffè’s test).