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. 2013 Jul 8;123(8):3243–3253. doi: 10.1172/JCI69134

Figure 4. Effects of ARHGDIA knockdown on RHO GTPase activity and podocyte migration in cultured human podocytes.

Figure 4

(A) Active GTP-bound RAC1 and CDC42 precipitated from podocytes transfected with scrambled (Scr) or ARHGDIA siRNA using a GST-PAK1 (CRIB) pulldown assay. Ponceau red staining at the top shows the GST proteins used. Compared with control podoctyes, podocytes transfected with ARHGDIA siRNA exhibited a significant increase in relative RAC1 and CDC42 (168% and 185%, respectively). The efficiency of knockdown by siRNA was confirmed by immunoblotting with an anti-ARHGDIA antibody (second to lowest panel). (B) Active GTP-bound RHOA precipitated from podocytes transfected with scrambled or ARHGDIA siRNA using a GST-rhotekin (RBD) pulldown assay. Cells transfected with scrambled control siRNA versus ARHGDIA siRNA exhibited no significant difference in relative RHOA activity. A and B represent 3 experiments each. (C–E) Quantification of RAC1 (C), CDC42 (D), and RHOA (E) in ARHGDIA-depleted cells compared with control cells. Error bars indicate the SEM for greater than 4 independent experiments. *P < 0.05; **P < 0.01; difference from proteins in control podocytes transfected with scrambled siRNA. (F) Effect of ARHGDIA knockdown on podocyte migration. Podocytes transfected with ARHGDIA siRNA exhibited more active migration compared with those transfected with scrambled siRNA. Increase in podocyte migration by ARHGDIA knockdown (red) in serum-induced podocytes was reduced by 2 different RAC1 inhibitors (green). Error bars are shown in only one direction for clarity and indicate SDs for more than 4 independent experiments.