Figure 8.

ASAP1 is a target of NAV-2729 but NAV-2729 may bind to additional proteins.A, NAV-2729 reduces ASAP1 in ruffles. U2OS cells were stained with Alexa568-phalloidin, as described in Figure 2D, and immunostained for ASAP1. Scale bars are 25 μms in length. B, fractional occupancy of ASAP1 in the cells shown in (A). The intensity of the ASAP1 signal at the cell edge was compared to total ASAP1. C, reduced expression of ASAP1 did not affect proliferation in RD or U2OS cells. Three diRNA targeting ASAP1 were used to knockdown ASAP1 for proliferation assays, and two were used to test NAV-2729 sensitivity. Relative cells mass was determined as in Figure 1C. Figures summarize two experiments. D, quantification of the number of actin fibers in RD cells and the cell surface area in U2OS cells plated and immunostained as described in Figure 2D. E, ASAP1 expression and NAV-2729 sensitivity are not correlated. Relative expression of ASAP1 was plotted against the EC50 for inhibition of cell proliferation. (Fig, 1C and Table 1). F, candidate approach identifies ROCK1, ROCK2, and Tiam1 as possible binding partners. Binding of proteins from lysates of RD cells to LUVs with and without NAV-2729 was determined as described in Experimental procedures. Each protein was measured in ≥3 experiments performed in duplicate. All data points were plotted. The data were analyzed by two-way ANOVA followed by Sidak’s multiple comparison test implemented in GraphPad Prism. G, validation of proteins identified in cellular thermal shift assay screen. Experiment performed as described in (F). Each protein was measured in ≥3 experiments performed in duplicate. A similar analysis was performed as described in (F). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. LUV, large unilamellar vesicle.