FIGURE 5.

MyoD-Y156 phosphorylation increases its stability by interfering with MAFbx/AT-1 binding. A, 10T1/2 cells transfected with either wild type or mutant MyoD (Y156F, S200G/Y156F, L164Q/Y156F) were extracted and analyzed by immunoblotting using anti-MyoD (C-20) antibody. The ratio of MyoD protein to α-tubulin is shown in the lower panel. B, 10T1/2 cells transfected with either wild type or mutant MyoD (Y156F, Y156E) were extracted and analyzed by immunoblotting using anti-MyoD (C-20) antibody. C, the Tyr-156 residue (*) and the interacting region (boldface line) of MAFbx/AT-1 in MyoD are conserved among several species. D, 10T1/2 cells were transfected with FLAG-MyoD and HA-MEKEE (ΔN) and separately with Myc₆-MAFbx/AT-1. After 24 h, each cell extract (Ext.) was prepared separately, mixed, and immunoprecipitated with anti-MyoD (C-20) antibody. The immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody. The results show the mean ± S.D. of triplicate experiments. *, p < 0.05; **, p < 0.01.