TRAF6 is required for the activation of NF-κB transcription factor in denervated skeletal muscle. (A) DNA-binding activity of NF-κB measured by EMSA in TA muscle of TRAF6^f/f and TRAF6^mko mice 10 d after denervation. A representative EMSA gel from three independent experiments is presented. (B) Quantification of fold change in DNA-binding activity of NF-κB in TA muscle of TRAF6^f/f (n = 6) and TRAF6^mko (n = 6) mice. (C) Fold change in NF-κB reporter gene activity (normalized using Renilla luciferase) in TA muscle of TRAF6^f/f and TRAF6^mko mice in response to denervation. (D) Western blot analyses of the phosphorylated and total form of p65 protein in TA muscle of TRAF6^f/f and TRAF6^mko mice. (E) Western blot analysis of Fn14 protein in TA muscle of TRAF6^f/f and TRAF6^mko mice 4 d after denervation. (F) TRAF6^+/+ and TRAF6^−/− MEF were treated with 100 ng/ml TWEAK for the indicated time intervals, and the activation of NF-κB was studied by EMSA. A representative EMSA gel from two independent experiments is presented. (G) Fold change in NF-κB reporter gene activity (normalized using Renilla luciferase) in TRAF6^+/+ and TRAF6^−/− MEFs measured after 24 h of 100 ng/ml TWEAK treatment. Error bars represent SD. D, denervated; S, sham-operated. *, P < 0.01 (values significantly different from denervated TA muscle of TRAF6^f/f mice).