FIGURE 2:

Differential expression of SNX9 affects the activation of RhoGTPases. (A, B) Active forms of the indicated Rho-family GTPases were pulled down using beads coupled to respective effector domains that can only bind the GTP-bound (i.e., active) form of the GTPases (see Supplemental Figure S2, A and B, and Materials and Methods). Bar chart shows quantification of active forms of RhoA, RhoC, Cdc42, and Rac1 in 231-siSNX9 (A) or 231-oxSNX9 (B) after normalization to respective control cells. n = 3–6; *p = 0.02. (C) Myosin light chain (MLC2) and cofilin are phosphorylated downstream of RhoA-ROCK activation. The bar chart compares the phosphorylation of MLC2 and cofilin in control and SNX9-depleted cells. n = 3; *p = 0.05. (D) Representative Western blot of His-SNX9 interaction with GST-Cdc42 or GST-RhoA in vitro. Before transferring to nitrocellulose membranes, protein loading was measured on Stain-Free gels (see Materials and Methods) to ensure that comparable amounts of GST- vs. GST-Cdc42 or GST-RhoA beads were used in each condition. Blot is representative of three independent experiments. (E, F) P_i production after GTP hydrolysis by RhoA (E) or Cdc42 (F) either alone or incubated with SNX9 and/or p50GAP. p50GAP alone was used as a positive control for P_i production by the GTPases. n = 4; ****p < 0.0001.