Fig 3.

Role of TRAF6 in the induction of ER stress-responsive genes in skeletal muscle upon fasting. Three-month-old TRAF6^f/f and TRAF6^mko mice were fasted for 24 h, and isolated GA muscles were used for biochemical analysis. (A) ATF4, CHOP, GRP94, GADD34, and PDI transcript levels were found to be significantly lower in fasted GA muscles of TRAF6^mko mice than in those of TRAF6^f/f mice. n = 4 in each group. Error bars represent SD. *, P < 0.05 (values significantly different from those for muscles of starved TRAF6^f/f mice). (B) Representative immunoblots demonstrating reduced protein levels of ATF4 and CHOP in TA muscles of starved TRAF6^mko mice compared with those of TRAF6^f/f mice. C2C12 myoblasts were differentiated into myotubes and starved for 3 and 6 h in PBS. U, unstarved; S, starved. (C) Representative immunoblots from two independent experiments performed in triplicate showing increased phosphorylation of eIF2α protein and elevated levels of ER stress-responsive proteins ATF3, ATF4, PDI, and CHOP in myotubes starved for 3 h or 6 h. Levels of MyHCf were reduced upon incubation of myotubes in PBS. (D) Splicing of XBP-1 increased upon fasting or the ER stressor tunicamycin in C2C12 myotubes measured by QRT-PCR assays using primers that detected both unspliced and spliced forms of XBP-1. uXBP-1, unspliced XBP-1; sXBP-1, spliced XBP-1.