FIGURE 6: p38β MAPK activates ULK1 in skeletal muscle cells in response to tumor. (A) LCM stimulates ULK1 phosphorylation on Ser-555 in myotubes through p38 MAPK. C2C12 myotubes were pretreated with DMSO (0.1%, vehicle), compound C (CC, 10 mM) or SB202190 (SB, 10 mM) for 30 min, and then treated with LCM for 1 h. ULK1 phosphorylation state on Ser-555 and AMPK phosphorylation on Thr-172 in cell lysate were analyzed by Western blotting. (B) LCM stimulates p38 MAPK interaction with ULK1 in myotubes. C2C12 myotubes were treated with LCM for 1 h. Cell lysate was subjected to immunoprecipitation with pre-immune IgG or p38 MAPK-specific antibody. Precipitate was analyzed by Western blotting for p38 MAPK and pSer-555 ULK1. (C) LCM stimulation of ULK1 phosphorylation on Ser-555 in myotubes is mediated by p38β MAPK specifically. C2C12 myoblasts were transfected with control, p38α MAPK or p38β MAPK-specific siRNA. After differentiation for 96 h, myotubes were treated with LCM or control medium for 1 h. Knockdown of p38 MAPKs and phosphorylation of ULK1 on Ser-555 were analyzed by Western blotting. (D) p38β MAPK is required for ULK1 activation in cachectic muscle of LLC tumor-bearing mice. Mice with muscle-specific knockout of p38β MAPK and control mice were implanted with LLC cells. Lysate of TA collected from these mice on day 21 was analyzed by Western blotting for ULK1 phosphorylation on Ser-555 and AMPK phosphorylation on Thr-172. Data were analyzed by one way AVOVA. * denotes a difference (p < 0.05).