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Fig. 3. Co-dependence of - and ß-Spectrin for proper protein localization. (A-I) Stage 15 embryos stained with polyclonal anti-ß-Spectrin antibody (A,D,G) or with monoclonal anti--Spectrin antibody (B,E,H). A merged composite image of - and ß-Spectrin staining for each genotype is shown in C, F and I. (A-C) An -spectrin heterozygous embryo. (A) ß-Spectrin protein localizes to the plasma membrane surrounding all CNS cells and to the axon scaffold (white brackets). (B) -Spectrin shows similar localization. (C) A merged image (yellow indicates colocalization). (D-F) A ß-spectrin (em6) mutant embryo. (D) ß-spectrin mutant embryos have significantly reduced ß-Spectrin protein levels. (E) -Spectrin protein levels are also reduced and are almost undetectable when confocal settings identical to those used for the image in B are applied, suggesting that ß-Spectrin is required to maintain normal levels of -Spectrin. When the Photo Multiplier Tube (PMT) gain is increased, low levels of -Spectrin can be seen at the plasma membrane (starred arrowheads). (F) A merged image showing no colocalization. (G-I) An -spectrin mutant embryo. (G) In -spectrin mutants, ß-Spectrin is redistributed to axons (white brackets). (H) -spectrin mutants have low levels of -Spectrin. When PMT gain is increased, residual -Spectrin can be seen at the plasma membrane (arrowheads) but not on axons (arrows). (I) A merged image of - and ß-Spectrin localization in an -spectrin mutant. Arrowheads depict cells in which - and ß-Spectrin colocalize (yellow). For all panels, the genotypes are listed on the left and antibodies are listed on top.

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