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. 2015 May 11;125(6):2375–2384. doi: 10.1172/JCI79504

Figure 6. Effects of Kank2 knockdown on RHO GTPase activity and podocyte migration in cultured podocytes.

Figure 6

(A) Active GTP-bound RHOA precipitated from cultured human podocytes transfected with scrambled (Scr) or KANK2 siRNA using a GST-rhotekin (RBD) pull-down (PD) assay. Ponceau red staining shows the GST proteins used. Compared with control podocytes, podocytes transfected with KANK2 siRNA exhibit a relative increase in RHOA. The efficiency of knockdown by siRNA was confirmed by immunoblotting with an anti-KANK2 antibody. (B) Active GTP-bound forms of RAC1 and CDC42 precipitated from podocytes transfected with scrambled or KANK2 siRNA using a GST-PAK1 (CRIB) pull-down assay. Cells transfected with scrambled control siRNA versus KANK2 siRNA exhibited no significant difference in relative RAC1 and CDC42 activity. Pull-down experiments are representative of more than 3 experiments. (C) Effect of Kank2 knockdown on podocyte migration. Cultured murine podocytes transfected with Kank2 siRNA (red) exhibited less active migration compared with those transfected with scrambled siRNA (black). Decrease in podocyte migration by Kank2 knockdown was rescued by overexpression of human wild-type KANK2 in podocytes (green) but not by overexpression of the KANK2 p.S181G variant (blue). Overexpression of p.S684F resulted in partial rescue, indicating that this mutation is potentially a hypomorphic mutation. Error bars are shown in only one direction for clarity, and the result shown is representative of 3 experiments.