Fig. 2. HSPG GAG chain synthesis is temporally regulated. (A) Western blots of staged embryonic extracts probed with 3G10 antisera against GAG chain stub epitopes generated by heparitinase III digestion. GAG-modified core proteins are undetectable in 0-3 hour embryo extracts but are seen in 2-4 and 4-7 hour samples. No signal was detected in the absence of heparitinase. LC, loading control (B,C) Staged embryonic extracts were probed with (B) anti-Dlp to detect Dlp and with (C) anti-Myc to detect Dally. matTub>Gal4 was used to drive UAS-Dlp or UAS-Dally expression maternally and embryonic extracts from the indicated stages resolved on reducing gels. Dlp was detected as a sharp band at 80 kDa, corresponding to full-length protein, and a heterogeneous band between 50 and 60 kDa, which represents a GAG-modified cleavage product. Low levels of full-length Dlp are seen at 0-3 hours, but GAG modifications are essentially undetectable until 2-4 hour and are dramatically upregulated in 4-7 hour embryos. (C) Full-length epitope-tagged Dally is visible as an 80 kDa doublet in 0-3 and 2-4 hour extracts. Significant levels of GAG modification are first apparent in 4-7 hour extracts as decreased mobility of the full-length protein (asterisk). The 65 kDa band is likely to result from processing by protein convertases, similar to vertebrate glypicans.