Figure 2
From: Impaired NEPHRIN localization in kidney organoids derived from nephrotic patient iPS cells
R460Q NEPHRIN mutant protein is localized on the surface in HEK293 cells. (a) Immunostaining of HEK293 cells overexpressing wild-type (WT), R460Q mutant (Patient 3), and E725D mutant (Patient 0) NEPHRIN proteins, using an antibody against the extracellular domain of NEPHRIN in the absence of detergent. Wheat germ agglutinin (WGA) binds to the surface glycoproteins. Scale bars, 25 µm. (b) Immunostaining of HEK293 cells overexpressing wild-type (WT), R460Q mutant (Patient 3), and E725D mutant (Patient 0) NEPHRIN proteins, using an antibody against the intracellular domain of NEPHRIN in the presence of detergent. Note the comparable expression of the WT and mutant NEPHRIN proteins. Scale bars, 25 µm. (c) Western blotting analysis of HEK293 cells overexpressing wild-type (WT), R460Q mutant (Patient 3), and E725D mutant (Patient 0) NEPHRIN. The upper band of NEPHRIN (*) is retained in the R460Q mutant, but is absent in the E725D mutant. Three R460Q mutant clones were analyzed to exclude clonal variability. The result for the E725D mutant is consistent with our previous report17. The results from the different parts of the same blot were combined, as depicted by the solid boxes.
