Figure 6.

Effect of Fgd5-binding compounds on Fgd5-Rac1 interactions. (a) Inhibition of Rac1-Fgd5 binding was performed by pull-down assay in the presence of potential Fgd5 GEF inhibitors. Fgd5 triple domain (5 μM final concentration) was preincubated with the 11 compounds selected for further analysis (20 μM final concentration) then added to tubes containing immobilized GST-Rac1 (0.5 ml final volume). This showed that Fgd5 triple domain interaction with Rac1 could be significantly inhibited by aurintricarboxylic acid (ATA), mitoxantrone (MX) and chelerythrine (CY). (b) Specificity of compound inhibitory effect was examined by preincubation of TrioN with Fgd5-binding compounds then adding to tubes containing immobilized GST-Rac1. Mitoxantrone also inhibits TrioN-Rac1 binding and thus is not a selective inhibitor. Sample immunoblots from binding assays are shown below the histograms. (a and b) Error bars = s.e.m.; n = 3; significance was calculated using an unpaired Students t-test (*, p < 0.05; **, p < 0.01). Compound abbreviations: see legend to Figure 5