Figure 1

HhNp-GFP is functional. (A) Scheme of HhF-GFP (middle) and HhN-GFP (bottom) fusion constructs and predicted processing as compared to wild-type HhF (top). HhF-GFP is predicted to be processed into HhNp-GFP. (B-D) HhNp-GFP (green, C) is expressed in posterior cells labeled by fluorescent protein dsRed (red, D) and secreted (B), similar to wild-type HhNp (A/P boundary marked by a solid white line). Scale bar: 5 μm (E) Western blot of salivary gland protein extracts labeled with anti-GFP (upper panel) or tubulin (lower panel). Upper panel: as negative controls we used extracts of wild-type w¹¹¹⁸ larvae (lane 1) and larvae expressing an untagged HhF (lane 5), CD8-GFP was used as a positive control (lane2). A single 46 kDa band is seen in the lane with HhN-GFP expressing larvae (lane 3) and in the lane loaded with extract of HhF-GFP expressing larvae, two bands of 70 kDa and 46 kDa are seen (lane 4; U: unprocessed full-length HhF-GFP, P: processed HhNp-GFP). Lower panel: the same blot was reprobed with anti-tubulin for loading control. (F-I) HhF expression in adult wings. (F) Wild-type wing. (G) Wing from HhF-GFP rescue of hh^GS1 mutant has a similar phenotype to wild-type. (H) As a positive control, untagged HhF is expressed with 71B-Gal4 resulting in merged veins L2 and L3. (I) Ectopic expression of HhF-GFP has a similar phenotype to untagged HhF.