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Figure 4


Fig. 4. βV spectrin binds to F-actin and forms a complex with {alpha}II spectrin and band 4.1 in vivo. (A) GST and GST-tagged βV spectrin fragments used in the binding experiments (Coomassie-blue staining following SDS-PAGE). (B,C) βV spectrin–band-4.1 interaction. The endogenous {alpha}II spectrin binds to GST-βVR29 (B) and is immunoprecipitated by the anti-βV spectrin antibody, but not by the preimmune serum or by protein G alone (C). (D) F-actin co-sedimentation assay. The soluble (S) and pellet (P) fractions obtained upon ultracentrifugation are indicated. GST-tagged βVCH is not recovered in the pellet fraction (lane 3). When the same amount of GST-tagged βVCH is incubated with F-actin at 37°C for 30 minutes (lanes 5 and 6), almost all GST-tagged βVCH is recovered with F-actin in the pellet fraction after ultracentrifugation (lane 6). GST alone was used as a negative control (lanes 7-10). (E,F) βV spectrin–band-4.1 interaction. (E) In vitro binding assay. GST-tagged βVCH directly interacts with the SAB domain of band 4.1. (F) Co-immunoprecipitation assay. The anti-βV spectrin antibody immunoprecipitates band 4.1 from lysates of P8 mouse organs of Corti.





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