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. 2020 Oct 6;295(50):16920–16928. doi: 10.1074/jbc.AC120.015204

Figure 1.

Figure 1.

In addition to its canonical effect on RhoA, PRG DH/PH activates Cdc42 and promotes filopodia formation. A, hypothetical effects of membrane-anchored RH-RhoGEF DH/PH catalytic modules. B, confocal images of PAE cells showing EGFP-RH-RhoGEF DH/PH-CAAX constructs (green) and their effects on F-actin (red). Zoomed-in areas are shown in the bottom row. Scale bar, 20 μm. C, graph shows the percentage of cells exhibiting filopodia-like structures. Each dot represents the means ± S.E. of at least 30 cells per experiment (n = 3). ***, p < 0.0001; n.s., no significance, one-way ANOVA followed Tukey. D, representative blot shows expression of EGFP-RH-RhoGEF DH/PH-CAAX constructs. E and F, activation of Cdc42 (E) and RhoA (F) by EGFP-RhoGEF DH/PH-CAAX constructs transfected into HEK293T cells was detected by PAK-CRIB and Rhotekin-RBD pulldown (PD), respectively. The graphs represent the means ± S.E. densitometric values (n = 3). **, p = 0.005; ***, p < 0.001; ****, p < 0.0001, one-way ANOVA followed Tukey. G, pulldown of active EGFP-RhoGEF DH/PH-CAAX constructs based on their affinity for nucleotide-free recombinant Cdc42-G15A (left panel) and RhoA-G17A (middle panel). Total cell lysates (TCL) are shown in the right panel. H, interaction between PRG DH/PH and Cdc42-T17N was assayed in transfected HEK293T cells subjected to pulldown assays (PD:GST). D–H, protein expression is confirmed in total cell lysates.