FIGURE 4.

Dynamin regulates Nephrin trafficking but does not physically interact with Nephrin. A, WT-Nephrin-transfected cells were utilized to visualize Nephrin trafficking upon stimulation with glucose or PS. Although WT-Dynamin overexpression did not impair Nephrin trafficking, K44A-Dynamin overexpression prevented Nephrin trafficking to intracellular compartments. The bottom panel shows bar graph analyses of WT-Nephrin and FM dye 4-64 co-localization in the presence of WT-Dynamin or K44A-Dynamin. FM dye 4-64 was utilized to stain plasma membranes. *, p < 0.05; ***, p < 0.001. B, immunoprecipitation experiments in FLAG-Nephrin-overexpressing cells were performed to determine whether Dynamin and Nephrin interact. FLAG-Pin1 was utilized as a negative control. Co-transfection of FLAG-Nephrin and GFP-Nck1 or GFP-Podocin was utilized as a positive control. Immunoprecipitation efficacy was verified by Western blot. A FLAG antibody was utilized to detect the FLAG epitope in eluates; the interaction with podocin and Nck1 was determined using a GFP antibody, and interaction with Dynamin was determined using a Dynamin-specific antibody. Western blot analysis indicates that endogenous Dynamin does not immunoprecipitates with FLAG-Nephrin. Six independent experiments were performed. IB, immunoblot.