Fig. 9

Islr stabilizes Dvl2 via antagonizing LC3-mediated autophagy. a Immunofluorescence analysis of GFP-LC3⁺ puncta in C2C12 cells transfected with GFP-LC3 and Dvl2-Flag plasmids, GFP-LC3 and Islr-HA plasmids, or GFP-LC3, Dvl2-Flag, and Islr-HA plasmids. N = 3 cell cultures in each group. Approximately, 20 cells total in each group. The numbers of GFP-LC3⁺ puncta per cell are shown on the right. b Western blot analysis of p62 and LC3II protein levels in shCtrl and shIslr C2C12 cells after 2 d in growth medium. c Immunofluorescence analysis of GFP-LC3⁺/Dvl2-Flag⁺ puncta in shCtrl and shIslr C2C12 cells transfected with GFP-LC3 and Dvl2-Flag plasmids. N = 3 cell cultures in each group. Approximately, 20 cells total in each group. The numbers of GFP-LC3⁺/Dvl2-Flag⁺ puncta per cell are shown on the right. d Immunofluorescence analysis of GFP-LC3⁺/Dvl2⁺ puncta in shCtrl and shIslr C2C12 cells treated with rapamycin (Rap) for 6 h. N = 3 cell cultures in each group. Approximately, 15 cells total in each group. The numbers of GFP-LC3⁺/Dvl2⁺ puncta per cell are shown on the right. e Immunofluorescence analysis of GFP-LC3⁺/Dvl2⁺ puncta in satellite cells from control, Islr cKO, Dact1 cKO, and Islr/Dact1 cKO mice treated with rapamycin (Rap) for 6 h. N = 3 cell cultures in each group. Approximately, 15 cells total in each group. The numbers of GFP-LC3⁺/Dvl2⁺ puncta per cell are shown on the right. Scale bars are all 25 μm. f Western blot analysis of Dvl2, Axin2, and Lef1 protein levels in primary myoblasts of control and Islr cKO mice incubated with BFA1 or DMSO. g Reciprocal co-immunoprecipitation analysis between GFP-tagged Islr and Flag-tagged Dvl2 in HEK293T cells. IB immunoblotting, IP immunoprecipitation. h Reciprocal co-immunoprecipitation analysis between Islr and Dvl2 in primary myoblasts cultured for 4 d in differentiation medium. i Western blot analysis of the ubiquitination level of Dvl2 in primary myoblasts of control and Islr cKO mice cultured for 4 d in differentiation medium. Error bars represent the means ± s.d. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; Student’s t test