Fig. 2. Molecular characterization of ß-spectrin mutants. (A) Schematic drawing of the 2291-amino acid large ß-Spectrin protein. The position of the S012 mutation is indicated. The bar indicates the part of the ß-Spectrin protein used for immunization. (B) Western blot of cell extracts derived from stage 15-17 embryos probed with a polyclonal antiserum generated against a ß-Spectrin fusion protein. First lane: wild type (wt), the 280 kDa large ß-Spectrin protein and a 200 kDa degradation product is visible. The 50 kDa band is a background band recognized by the polyclonal antibody. Mutant ß-spectrin embryos were selected using GFP-carrying balancer chromosomes. The genotype is indicated. In em6, em15, em21, G113, E292, L105 and M046 hemizygous embryos, truncated ß-Spectrin proteins are detected. E175, H127 and S012 hemizygous-mutant embryos lack detectable ß-Spectrin expression. (C) The same membrane was probed for expression of -Spectrin. Notice the strong reduction of -Spectrin expression in all genotypes, regardless of whether residual ß-Spectrin protein can still be detected or not. (D) Coomassie staining of same membrane to demonstrate equal loading. ABD, actin-binding domain; SR, spectrin repeats; Ank, ankykrin domain; PH, pleckstrin homology domain.