Figure 3.

Generation of wrd loss-of-function mutants. a, Western blot confirming absence of Wrd protein in excision mutants. Genotypes are as follows: wild type, elav Gal4/+; wrd¹⁸⁹/Df(3R)DG4, Df(3R)189/Df(3R)DG4; and wrd¹⁰⁴ hz, wrd¹⁰⁴/wrd¹⁰⁴. α-Wrd reveals three bands (arrows) in wild-type animals, consistent with the presence of multiple wrd splice forms. These bands are absent in the lane for wrd¹⁸⁹/Df(3R)DG4, a definitive null lacking the wrd gene, showing that the antibody specifically recognizes Wrd protein. The Wrd bands are also absent in the wrd¹⁰⁴ homozygote, which is therefore a protein null. Bottom half of blot shows a nonspecific background band, here shown as a loading control. b, Representative neuropil staining for Wrd in wild-type animals, wrd¹⁰⁴ mutants, and wrd¹⁸⁹ hz [Df(3R)189/Df(3R)189]. Animals were costained for Wrd and Bruchpilot, an active zone marker with strong neuropil staining. Bruchpilot staining is shown in insets. Absence of Wrd staining in wrd¹⁰⁴ mutants and wrd¹⁸⁹ hz demonstrates specificity of staining at synapses in neuropil. Remaining staining outside of neuropil represents background, because it persists in wrd¹⁸⁹ hz, a definitive null. c, Representative synaptic staining for Wrd at the NMJ on muscle 4 of wild-type animals, wrd¹⁰⁴ mutants, and wrd¹⁸⁹ hz. The NMJ is stained for Wrd as well as HRP to delineate the synaptic terminal. Whereas Wrd staining is present at the NMJ in wild-type animals, it is essentially absent in the wrd¹⁰⁴ mutant and the wrd¹⁸⁹ hz, demonstrating specificity of Wrd staining at NMJ.