Fig. 4. Impaired chondroitin sulfation and altered aggrecan metabolism in Jaws^-/- limbs. (A) CS immunostaining in hindlimb digits (E14.5). Higher-magnification views (top) reveal sparse irregular staining in the Jaws^-/- joint. (B,C) Fluorophore-assisted carbohydrate electrophoresis analysis of chondroitin sulfate (CS, B) and heparan sulfate (HS, C) in E14.5 limbs. The relative proportions and percentages of disaccharides generated with the glycosaminoglycan-specific lysases are shown. For HS, no disaccharides containing 2-sulfated uronic acid (UA) or N,6-sulfated GlcN were detected. galNAc, N-acetylgalactosamine; C0S, UA-galNAc; C4S, UA-galNAc4S; C6S, UA-galNAc6S; glcNAc, N-acetylglucosamine; GlcN, UA-glcNAc; GlcNS, UA-glcNS; GlcN(6S), UA-glcNAc6S. ^*P<0.025; P<0.0001, Jaws^-/- versus wild type percentage (n=5 pairs per genotype). (D) Comparable amounts (mean and s.d.) of total glycosaminoglycans in wild type and Jaws^-/- hindlimbs, normalized to wet tissue weight. Similar results were obtained for forelimbs. n.d., not determined; HA, hyaluronan. (E) Aggrecan immunostaining in the proximal end of the humerus (E14.5). (F) Anti-CDAGWL immunoblots of E14.5 limb protein extracts, showing less full-length aggrecan (arrow, 350 kDa) and fewer aggrecan cleavage fragments (bracket) in Jaws^-/- cartilage. Successive nondissociative extractions (lanes 1 and 2; arrowhead indicates a 65 kDa proteolytic fragment) were followed by one dissociative extraction (lane 3). HL, hindlimbs; FL, forelimbs.