Figure 5

Western blot analysis, qRT-PCR and ELISA show increased levels of NFκB–mediated inflammation starting at postnatal week 1 and continuing through development. (A) Western blot of total and phosphorylated p65 reveals that DyW mice had elevated levels of NF-κB signaling beginning at postnatal week 1 and continuing throughout development. (B) TNF-α gene analysis shows that DyW muscles had significantly elevated expression at week 1 (P < 0.01), week 3 (P < 0.0001) and week 4 (P < 0.05 by two-way ANOVA (n = 5) for all groups). (C) Gene expression analysis of MCP-1, reveals that DyW and WT mice had comparable expression levels until postnatal week 4, at which point the DyW MCP-1 level became significantly elevated (P < 0.0005 by two-way ANOVA (n = 5)). (D) Osteopontin (OPN) was also highly upregulated (>25 –fold) at every time point (P < 0.0005 by two-way ANVOA (n = 5) for all groups). (E) ELISA of TNF-α shows elevated expression levels in DyW mice starting at week 2 (P < 0.05 for week 2, P < 0.0001 for weeks 3 and 4, both by two-way ANOVA (n = 3)). (F) MCP-1 was also found to be upregulated at weeks 2, 3 and 4 (P < 0.0001 by two-way ANOVA (n = 3) for all groups). (G) Osteopontin protein was also seen to be upregulated at each of the four time points (P < 0.0001 by two-way ANOVA (n = 5)), with the largest increases seen in the first 3 weeks. Asterisks indicate statistical significance between age-matched DyW and WT, hash tags indicate significant changes within the DyW group and Deltas indicate significant changes within the WT group. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P< 0.0001. These statistical representations also apply other symbols (Δ, #).