Fig. 2. Shot type A and B N-terminal domains associate with F-actin. (A) NIH3T3 fibroblasts transfected with type A, B, or C Shot N-terminal domains fused to GFP. CH, calponin homology domain. F-actin (red) is visualized using Cy3-conjugated phalloidin. Type A- and B-GFP fusions (green) co-localize with F-actin in the cortical region (arrowhead) and stress fibers (arrows) of transfected cells, but type C-GFP fusions (green) do not. Yellow (see merge panels) indicates the overlap between the F-actin and GFP distributions. Images are 1 µm confocal sections. Scale bar: 10 µm. (B) The in vitro actin-binding properties of Shot N-terminal domains. GST (Glutathione S-Transferase) fusions (2 µM) with either the second calponin homology domain (CH2) or the two calponin homology domains (CH1 + CH2) were incubated with F-actin (19.2 µM) and the samples separated into supernatant (S) or pellet (P) fractions by 1 hour of centrifugation at 150,000 g. The proteins in each fraction were then electrophoretically separated. The positions in the gel of the (CH1 + CH2)-GST fusion (open arrowhead), the CH2-GST-fusion (black arrowhead) and actin (arrow) are indicated. In the absence of F-actin, (lanes 1, 2, 7, 8), little fusion protein pellets. Little CH2-GST fusion is associated with F-actin (lanes 4, 6); most remains in supernatant fractions (lanes 3, 5). As CH2-GST is not well resolved from actin, the samples in lanes 3-4 were transferred electrophoretically to Immobilon P membranes (Millipore) and probed with anti-GST (lanes 5-6) to determine how much CH2 is associated with F-actin. CH2-GST largely remains in the supernatant after F-actin is pelleted (compare lane 5 with lane 6). By contrast, most of the (CH1+CH2)-GST fusion pellets with F-actin under these conditions (compare lane 9 with lane 10). (C) Scatchard plot of the binding reaction between the (CH1+CH2)-GST fusion and F-actin. Binding of the (CH1+CH2)-GST fusion to F-actin (4 µM) was measured at fusion protein concentrations of 100, 200, 300 and 400 nM. The relative amounts of proteins in the supernatant and pellet were determined by quantitative densitometry of silver-stained SDS-polyacrylamide gels. Three data sets were averaged; individual values differed from the average by no more than 9%. Vertical axis, (CH1+CH2)–GST bound/free (CH1+CH2)–GST (µM^–1); horizontal axis, bound (CH1+CH2)–GST/F-actin.