Fig. 5.

TM4SF10 colocalizes with nephrin and is associated with changes in nephrin abundance and phosphorylation in podocytes. A–C: immunohistochemistry for F-actin (Phalloidin) comparing a podocyte with and without TM4SF10-GFP expression. Cells expressing TM4SF10-GFP cells had more circumferential bands (B, arrows) of F-actin, whereas cells without TM4SF10-GFP had a more typical stress fiber appearance of F-actin cross striations. D–G: colocalization of TM4SF10-GFP and nephrin in podocytes. E–G: higher magnification of boxed region in D showing partial colocalization of nephrin and TM4SF10-GFP in fine, distal cell extension. H: Western blot of podocyte nephrin expression following overexpression or knockdown of TM4SF10-GFP using a TM4SF10-specific short hairpin RNA (shRNA). The high-molecular-weight forms of nephrin at 175 and 185 kDa are noted with arrows. Efficiency of shRNA knockdown of TM4SF10 was confirmed by Western blot with anti-GFP antibodies and Tubulin was used as a loading control. I: Western blot of total nephrin abundance and presence of nephrin tyrosine¹²¹⁷ phosphorylation (pY1217) with and without TM4SF10-GFP overexpression and with and without preincubation with pervanadate to block tyrosine phosphatase activity.