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Fig. 9. The F-actin bundling activity of the IMD of MIM-B is inhibited by Rac. (A) Protein used in this assay. Recombinant His-tagged IMD and GTPase mutants were purified to homogeneity and analysed on SDS-PAGE followed by Coomassie Blue staining. Position of molecular size marker in kDa is indicated. (B) In this representative experiment, 5 µM His-tagged IMD (see Fig. 9A) were pre-incubated with various concentrations of recombinant N17-Rac or L61-Cdc42 (see Fig. 9A) before addition of 5 µM F-actin. F-actin bundling was determined as described in Fig. 7. Pellet fractions were analysed by SDS-PAGE and the band intensity of actin was measured densitometrically. Percentage of F-actin bundled was normalised by F-actin bundled in the absence of IMD and expressed as percentage of bundling inhibition.





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