Fig. 5. CMS binding to F-actin in whole-cell extracts mirrors the binding measured with purified components. Cell lysates from 293T cells expressing the FLAG-tagged CMS constructs depicted in the scheme were incubated with exogenous F-actin and tested for their ability to co-sediment (lanes 3 to 12). F-actin was sedimented by centrifugation at 100,000 g. The soluble (S) and actin-bound fractions (P) were resolved by SDS-PAGE and analyzed by western blot for the presence of F-actin and CMS. Lanes 1 and 2 represent negative controls without F-actin. The CC domain and PR region deletion mutants of CMS show a diminished F-actin binding activity, similarly to the results of GST pull-downs assays (Fig. 3). F-actin-binding was scored: +++, strong; ++, medium; +, low; –, no binding.