Fig. 6. Histochemistry for chondroitin 4 epitopes and HS in adult female and L3 larvae. Sections from adult female and L3 larvae were treated with anti-C4S or anti-HS monoclonal antibodies, as described in Materials and methods. e, oocytes; i, intestine; n, nurse cells; sgl, salivary gland; pv, proventriculum; b, brain hemisphere; f, fat body; s, stomach. Control was obtained omitting the primary antibody. From: Biosynthesis and metabolism of sulfated glycosaminoglycans during Drosophila melanogaster development Glycobiology. 2004;14(6):529-536. doi:10.1093/glycob/cwh070 Glycobiology | Glycobiology vol. 14 no. 6 © Oxford University Press 2004; all rights reserved.

Fig. 5. Strong anion-exchange HPLC analysis of the disaccharides formed by a specific lyase on the glycans obtained from different stages of development. A mixture of standard chondroitin disaccharides and the disaccharides formed by exhaustive action of chondroitinase ABC on the glycans extracted from embryos, L1, L2, and L3, were applied to a 250 mm × 4.6 mm Spherisorb-SAX column linked to an HPLC system. The column was eluted with a linear gradient of NaCl, as described in Materials and methods. The chondroitin disaccharide standards used were: ΔDi-0S, ΔUA-1→3-GalNAc; ΔDi-4S, ΔUA-1→3-GalNAc(4SO₄); ΔDi-6S, ΔUA-1→3-GalNAc(6SO₄). Arrows indicate the position of elution of the standards disaccharides. The results were reproduced in duplicate experiments. From: Biosynthesis and metabolism of sulfated glycosaminoglycans during Drosophila melanogaster development Glycobiology. 2004;14(6):529-536. doi:10.1093/glycob/cwh070 Glycobiology | Glycobiology vol. 14 no. 6 © Oxford University Press 2004; all rights reserved.

Fig. 4. Biosynthetic activity in embryos of different ages Fig. 4. Biosynthetic activity in embryos of different ages. Adult flies were labeled with ³⁵S-sulfate for 24 h, as described in Materials and methods. (A) Embryos at 2, 4, 6, 8, 10, and 12 h of age were collected; the ³⁵S-sulfate-glycans were extracted by proteolytic digestion and ethanol precipitation. Low-molecular-weight contaminants were washed out by paper chromatography and the amount of ³⁵S-sulfate-glycans recovered in the embryos counted and estimated as cpm/mg dry tissue (mean ± SD, n = 3). (B) Agarose gel electrophoresis autoradiography of the ³⁵S-sulfate-glycans extracted from embryos at different ages. Similar amount of radioactivity in terms of cpm/mg dry tissue of each glycan was applied to the agarose gel. From: Biosynthesis and metabolism of sulfated glycosaminoglycans during Drosophila melanogaster development Glycobiology. 2004;14(6):529-536. doi:10.1093/glycob/cwh070 Glycobiology | Glycobiology vol. 14 no. 6 © Oxford University Press 2004; all rights reserved.

Fig. 3. Pulse-chase of ³⁵S-sulfate-glycans during embryonic and larval development. (A) Adult flies (male and female) were fed for 72 h with Na₂³⁵SO₄. During this period ³⁵S-sulfate-labeled embryos were collected in acetone at 24-h intervals and ³⁵S-sulfate-labeled adult flies collected in acetone at the end of the 72-h period. To obtain ³⁵S-sulfate-labeled larvae, adult flies were fed with the feeding mixture for 72 h as described. The ³⁵S-sulfate-labeled embryos from the first 48 h were removed and the embryos from the last 24 h transferred to a medium containing the feeding mixture without Na₂³⁵SO₄. L1, L2, and L3 ³⁵S-sulfate-labeled larvae were collected in acetone after 24 h, 72 h, or 144 h, respectively. ³⁵S-sulfate-glycans from adult females, embryos, and larvae were extracted and the amount of incorporation in the different stages of development estimated by cpm/mg dry tissue (mean ± SD, n = 3), as described. The content of ³⁵S-sulfate-glycans obtained from embryo are significantly different from those of adult female (^*p < 0.01). Similarly, the content of ³⁵S-sulfate-glycans from larvae (L1, L2, and L3) are significantly different from those of embryo (^**p < 0.05). (B) ³⁵S-sulfate-glycans extracted from adult female, embryos, L1, L2, and L3 were analyzed by gel electrophoresis. (C) The identity of the glycans was confirmed by agarose gel electrophoresis before (−) or after (+) incubation with chondroitinase-AC and -ABC and deaminative cleavage with nitrous acid. From: Biosynthesis and metabolism of sulfated glycosaminoglycans during Drosophila melanogaster development Glycobiology. 2004;14(6):529-536. doi:10.1093/glycob/cwh070 Glycobiology | Glycobiology vol. 14 no. 6 © Oxford University Press 2004; all rights reserved.

Fig. 2. Time course of ³⁵S-sulfate incorporation into GAGs in male and female. (A) Adult male and female flies were fed separately with Na₂³⁵SO₄ for different times. ³⁵S-sulfate-glycans were extracted from the flies obtained from each feeding time, and the amount of radioactivity incorporated was estimated by cpm/mg dry tissue, as described in the legend of Figure 1. (B) ³⁵S-sulfate-glycans extracted from the flies from different feeding times were analyzed by agarose gel electrophoresis, as described in Materials and methods. The electrophoretic mobility of standard CS and HS are indicated. From: Biosynthesis and metabolism of sulfated glycosaminoglycans during Drosophila melanogaster development Glycobiology. 2004;14(6):529-536. doi:10.1093/glycob/cwh070 Glycobiology | Glycobiology vol. 14 no. 6 © Oxford University Press 2004; all rights reserved.

Fig. 1. Incorporation of ³⁵S-sulfate into GAGs in adult male and female D. melanogaster and characterization of the ³⁵S-sulfate-glycans. Adult flies (male and female) were fed with Na₂³⁵SO₄ for 72 h, as described in Materials and methods. (A) After the feeding period, ³⁵S-sulfate-glycans were extracted and the amount of ³⁵S-sulfate incorporated was estimated by cpm/mg dry tissue (mean ± SD, n = 3), after washing out low-molecular-weigh contaminants by paper chromatography, as described in Materials and methods. (B) ³⁵S-sulfate-glycans (∼20,000 cpm) from adult flies and a mixture of standard GAGs containing CS, DS, and HS (20 µg each) were applied to a 0.5% agarose gel in 0.05 M 1,3 diaminopropane/acetate (pH 9.0), and run for 1 h at 110 mV. After electrophoresis, the glycans were fixed and stained, as described in Materials and methods. The radioactive bands corresponding to the ³⁵S-sulfate-glycans were detected by autoradiography of the stained gel. (C) ³⁵S-sulfate-glycans were identified by agarose gel electrophoresis before (−) or after (+) incubation with chondroitinase (Chase) AC or ABC or deaminative cleavage with nitrous acid. M, adult male; F, adult female. From: Biosynthesis and metabolism of sulfated glycosaminoglycans during Drosophila melanogaster development Glycobiology. 2004;14(6):529-536. doi:10.1093/glycob/cwh070 Glycobiology | Glycobiology vol. 14 no. 6 © Oxford University Press 2004; all rights reserved.