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. 2005 Jan 6;24(2):240–250. doi: 10.1038/sj.emboj.7600535

Figure 4.

Figure 4

In vitro characterisation of the IMD of IRSp53 and interaction with F-actin. (A) SDS–polyacrylamide gel electrophoresis (SDS–PAGE) of purified IMDwt (wt) and IMDC230A (C230A) analysed under reducing (+DTT) and nonreducing (−DTT) conditions after heat denaturation. All gels in this figure were stained with Coomassie blue and positions of molecular weight markers are indicated in units of 103 Da. (B) Sedimentation coefficient distributions of IMDwt and IMDmut. The main peak for both is at 3.25s, while some higher order aggregate is observed for the mutant at 4.5s. (C) Circular dichroism spectroscopy of wild-type (thick line) and mutant (thin line) forms of the IMD. (D) High-speed cosedimentation assay of the interaction of wild-type (wt) and mutant IMD (mut) with F-actin (2.5 μM). Pellet fractions were analysed by SDS–PAGE and the band intensity of the IMD was measured densitometrically and normalised to actin. (E) Low-speed co-sedimentation assay of F-actin bundling. Pellet (P) and supernatant (S) fractions were analysed by SDS–PAGE. (F) Fluorescence microscopy-based F-actin-bundling assay. Cy3-labelled F-actin (1 μM) was incubated with 5 μM wt or mut IMD and imaged using a fluorescence microscope (scale bar=20 μm).