Cells were transferred into six-well plates (Thermo Fisher Scientific) for immunoblotting experiments or 35-mm glass-bottom dishes (Mattek) for imaging experiments 1 d before transient transfection. Cell confluency at the point of transfection was ∼60–80%. For TIRF imaging, to enhance even attachment of cells to the surface, 35-mm glass-bottom dishes (Mattek) were coated with polylysine (by incubating with 0.1% l -lysine [Sigma] solution for 30 min at room temperature followed by two washes with growth media) before cells were applied.
Effectene (QIAGEN) was used for transient transfection according to the manufacturer’s protocol with 0.4 μg of DNA for each sample. For coexpression of Src-FKBP, nephrin-FRB, and Nck1 (or engineered Nck1 proteins), 0.1, 0.2, and 0.1 μg of DNA were used, respectively. When either Src-FKBP or Nck1 was not expressed, empty pcDNA vector (Invitrogen) was used to match the total DNA amount. Samples were incubated for 27 h at 37°C prior to experiments to ensure sufficient expression and membrane localization of nephrin-FRB. Experiments were done using cells incubated no longer than 35 h after transfection to avoid cell damage from unregulated Src kinase activity and overexpressed Nck1.
Effectene (QIAGEN) was used for transient transfection according to the manufacturer’s protocol with 0.4 μg of DNA for each sample. For coexpression of Src-FKBP, nephrin-FRB, and Nck1 (or engineered Nck1 proteins), 0.1, 0.2, and 0.1 μg of DNA were used, respectively. When either Src-FKBP or Nck1 was not expressed, empty pcDNA vector (Invitrogen) was used to match the total DNA amount. Samples were incubated for 27 h at 37°C prior to experiments to ensure sufficient expression and membrane localization of nephrin-FRB. Experiments were done using cells incubated no longer than 35 h after transfection to avoid cell damage from unregulated Src kinase activity and overexpressed Nck1.