Figure 4. The phosphorylation of... | Rockefeller University Press

Figure 4.

$The phosphorylation of Dyn2-S848 promotes endocytosis. (A and B) Subcellular distribution of endogenous GLUT4 in C2C12 myotubes expressing different Dyn2 mutants. Differentiated C2C12 myotubes were infected with lentiviruses to express indicated HA-Dyn2 and then subjected to subcellular fractionation to determine the distribution of endogenous GLUT4 by Western blotting. The markers for heavy membrane (Na+/K+ ATPase, plasma membrane), light membrane (Golgin-97, trans-Golgi), and cytosol (tubulin) were used to validate this assay. The ratio of GLUT4 in heavy membrane fraction was quantified and shown in B (n = 4 experimental replicates). Molecular weight is in kD. (C–E) Effects of Dyn2-S848 mutations on the insulin-regulated GLUT4-HA-GFP distribution. L6 myoblasts cotransfected with GLUT4-HA-GFP and Dyn2-mCherry mutants were subjected to growth or serum-free medium for 2 h. The cartoons illustrate the expected location of GLUT4-HA-GFP under indicated conditions; with or without receptor tyrosin kinase (RTK) signaling activation. After immunofluorescent staining with anti-HA antibody without permeabilization and imaging by confocal microscopy, the relative amount of surface GLUT4-HA-GFP was quantified by the fluorescent intensity of HA signaling in blue divided by the total GLUT4-HA-GFP in green as shown in E (n ≥ 27 cells from three independent repeats). Data are shown as average ± SD and analyzed with one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData F4.$

The phosphorylation of Dyn2-S848 promotes endocytosis. (A and B) Subcellular distribution of endogenous GLUT4 in C2C12 myotubes expressing different Dyn2 mutants. Differentiated C2C12 myotubes were infected with lentiviruses to express indicated HA-Dyn2 and then subjected to subcellular fractionation to determine the distribution of endogenous GLUT4 by Western blotting. The markers for heavy membrane (Na⁺/K⁺ ATPase, plasma membrane), light membrane (Golgin-97, trans-Golgi), and cytosol (tubulin) were used to validate this assay. The ratio of GLUT4 in heavy membrane fraction was quantified and shown in B (n = 4 experimental replicates). Molecular weight is in kD. (C–E) Effects of Dyn2-S848 mutations on the insulin-regulated GLUT4-HA-GFP distribution. L6 myoblasts cotransfected with GLUT4-HA-GFP and Dyn2-mCherry mutants were subjected to growth or serum-free medium for 2 h. The cartoons illustrate the expected location of GLUT4-HA-GFP under indicated conditions; with or without receptor tyrosin kinase (RTK) signaling activation. After immunofluorescent staining with anti-HA antibody without permeabilization and imaging by confocal microscopy, the relative amount of surface GLUT4-HA-GFP was quantified by the fluorescent intensity of HA signaling in blue divided by the total GLUT4-HA-GFP in green as shown in E (n ≥ 27 cells from three independent repeats). Data are shown as average ± SD and analyzed with one-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available for this figure: SourceData F4.