Podocin^{TVV339,340,341} regulates the turnover of podocin through a lipid raft-independent mechanism.

<p>A. Differentiated podocytes stably expressing Flag-tagged podocin, podocin^1–285 or podocin^{TVV339,340,341AAA} 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. Podocin^1–285 and podocin^{TVV339,340,341AAA} were shown to be more stable than podocin wild type, consistent with a regulatory role of podocin^{TVV339,340,341} 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 podocin^{TVV339,340,341} 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 podocin^{TVV339,340,341AAA} were detected in DRM. C. Graphical representation of the structure prediction analysis of podocin using the I-Tasser algorithm revealed exposed position of podocin^{TVV339, 340,341}.