Figure 3

The podocin short isoform is retained in the ER and is found in the detergent resistant membrane fraction. A) PCR with podocin specific primers yields products for both isoforms from a human kidney cDNA library. B) Immunofluorescence staining of the canonical and the short isoform in Hela cells transfected with the corresponding constructs show that only the canonical isoform reaches the plasma membrane (arrowhead in the enlarged part of the image). The short isoform is retained in the endoplasmic reticulum. Scale bar = 10 μm. C) DRM association of both podocin isoforms. HEK293T cells expressing the respective V5-tagged podocin isoform were lysed in 1% TX-100 on ice and subjected to sucrose density gradient centrifugation. Fractions 1–7 were collected from the top and analyzed by Western blot. Both isoforms fractionate into the DRMs (fractions 1 and 2, as identified by flotillin staining). Antibodies against the transferrin receptor (TfR) and Flotillin-2 were used as markers for the Triton soluble and insoluble fractions, respectively.