A Novel Domain Regulating Degradation of the Glomerular Slit Diaphragm Protein Podocin in Cell Culture Systems
Figure 5
PodocinTVV339,340,341 regulates the turnover of podocin through a lipid raft-independent mechanism.
A. Differentiated podocytes stably expressing Flag-tagged podocin, podocin1–285 or podocinTVV339,340,341AAA were exposed to the translation inhibitor cycloheximide for the times as indicated and analyzed per western blot using anti-Flag antibody (a). Actin levels detected by anti-actin antibody served as loading control. Podocin1–285 and podocinTVV339,340,341AAA were shown to be more stable than podocin wild type, consistent with a regulatory role of podocinTVV339,340,341 in its degradation. (b) Summarizes the results of three experiments. Podocin levels were normalized to actin levels. B. (a) shows a schematic comparison between the PHB-domain proteins podocin and stomatin. Umlauf et al. proved a motif partially overlapping with podocinTVV339,340,341 to play a crucial role in lipid raft binding. (b) HEK293T cells were transfected with the plasmids as indicated, lysed in 1% TX-100 on ice and subjected to flotation gradient centrifugation to prepare detergent-resistant membranes (DRM). In contrast to the control protein transferrin receptor, both podocin wild type and podocinTVV339,340,341AAA were detected in DRM. C. Graphical representation of the structure prediction analysis of podocin using the I-Tasser algorithm revealed exposed position of podocinTVV339, 340,341.