Figure 3.

INF2 preserves peripheral membrane trafficking of SD proteins by opposing Rho/mDia activity. Cultured podocytes (undifferentiated) were transfected with INF2-targeting siRNA duplex (INF2 KD) or universal negative control RNA duplex (control). Forty-eight hours later, the cells were cotransfected with plasmids encoding HA-podocin or nephrin. (A) HA (newly synthesized podocin, green) was costained with cortactin (red), and the lamellipodial distribution of podocin was compared in cells with or without INF2 KD. (B) The distribution of newly synthesized nephrin (red) and the association with actin filaments (blue) were compared in cells with or without INF2 KD. (C and D) Cells with INF2 KD were cotransfected with DN RhoA or DN mDia1 or were treated with y27632, a Rock inhibitor. (C) The distribution of podocin and the association with F-actin are illustrated by immunofluorescent stain and phalloidin stain. (D) Surface biotinylation was used to measure the surface membrane trafficking of nephrin. The cytosolic (C) and surface (S) nephrin was measured by Western blotting, and the membrane trafficking of nephrin was quantified as the fraction of biotinylated nephrin (S/S+C)%. HSP 70 and Na+/K+ ATPase were used as controls for unbiotinylated and surface-biotinylated proteins, respectively.