Figure 8.

4.1R forms carrying exon 17b associate with spectrin and actin and induce fordin–actin–4.1R ternary complex formation. A, association of endogenous 17b-containing 4.1R forms, actin, and spectrin was analyzed in an immunoprecipitation assay. Left, MDCK cell lysates were immunoprecipitated with a control MIgG, anti-β-actin, anti-α-spectrin, anti-ex17b Ab, or a control RIgG and examined with their respective antibodies for the presence of the precipitated protein. Right, MDCK lysates precipitated with MIgG, anti-β-actin, anti-βII-spectrin, anti-ex17b Ab, or RIgG and immunoblotted with an anti-βII-spectrin Ab for its presence in each precipitate. Molecular mass markers (kDa) are provided. B, schematic diagram of GST–4.1R–exon 16–17−17b constructs carrying various exon compositions used for the sedimentation assays. C, Coomassie Blue-stained gel of bacterially-expressed GST fusion proteins of different combinations of exon 16–17−17b peptides. Arrowhead, degradation product in purified GST–Ex17/17b fusion. Molecular mass markers (kDa) are provided. D, co-sedimentation assays using GST–4.1R peptides, rat brain fodrin, and F-actin followed by separation of supernatant (S) and pellet (P) fractions by ultracentrifugation and analysis by SDS-PAGE. Three independent experiments were performed. E, graphic presentation of the results obtained in D using National Institutes of Health Image software for quantitation of the fodrin band present in the pellet fraction.