Fig. 6.

Surface levels of Robo are increased and mislocalized in kuz mutants. (A-F) Stage 15 kuz/+ heteroygotes (A-C) or kuz mutant (D-F) embryos were dissected and stained live to reveal Robo surface expression. Anterior is up. Embryos were stained together on the same slide and micrographs were generated using identical confocal settings. (A-C) A kuz/+ heterozygous embryo stained with anti-HRP to reveal all axons (A) and with anti-Robo to visualize Robo distribution (B). Robo is enriched on longitudinal connectives and almost absent from axon commissures (compare B and C). (D-F) A kuz mutant embryo exhibits increased levels of Robo expression (compare E and B) and Robo protein is no longer restricted from the axon commissures (E,F). (G) A western blot from embryonic extracts of the indicated genotypes reveals increased Robo expression levels. Anti-tubulin was used as a loading control. (H) Quantification of Robo fluorescence intensity reveals a significant increase in Robo surface expression in kuz mutants. Each bar in the histogram represents an individual embryo. Fluorescence intensity was calculated as previously reported (Yang and Bashaw, 2006).