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Files in this Data Supplement:
Fig. S1. Disaccharide composition analysis of chondroitin sulfate GAG chains. Chondroitin sulfate GAG chains were digested with chondroitinase ABC (cABC), followed by fluorescent labeling with 2AB. Chondroitin sulfate GAG disaccharides were separated by anion-exchange HPLC on an amine-bound silica PA03 column using a gradient as indicated by the dashed line. The eluates were monitored by fluorescence intensity with excitation and emission wavelengths of 330 and 420 nm, respectively. The elution positions of the authentic 2AB-derivatized disaccharide are indicated by numbered arrows. Arrow 1, 3′-Sialy-N-acetyllactosamine; arrow 2, ΔDi-0S; arrow 3, ΔDi-6S; arrow 4, ΔDi-4S; arrow 5, ΔDi-2,6S; arrow 6, ΔDi-4,6S; arrow 7, ΔDi-2,4,6S. The peaks labeled with asterisks are derived from the 2AB-labeling reagent and/or unidentified impurities.
Fig. S2. Cerebral cortical neurons were repelled by CS-A as well as CSPG. Axonal guidance spot assays were performed with mouse cerebral cortical neurons. (A) Control, (B) CSPG, (C) CS-A and (D) CS-C. Cortical neurons were repelled by CS-A, but not by CS-C, similarly to CGNs.
Fig. S3. Increased production of CSPGs by reactive astrocytes is responsible for reduced neuronal growth. (A) Conditioned medium derived from 5 day astrocyte cultures with or without TGFβ1 were treated with chondroitinase ABC and subjected to immunoblot with CS-56. Immunoreactivity of CS-56 disappeared in response to chondroitinase ABC treatment of conditioned medium. (B) Conditioned medium derived from 5 day astrocyte cultures with or without TGFβ1 were separated on SDS-PAGE under non-reducing and reducing conditions and subjected to immunoblot with CS-56. CS-56-positive bands migrated more slowly and diffusely under non-reducing conditions. (C) Distribution of CSPGs on untreated (left) and TGFβ1-treated (right) astrocytes. Confluent monolayers of astrocytes were treated with TGFβ1 (10 ng/ml) in serum-free medium for 3 days. Living astrocytes were incubated with CS-56, followed by incubation with a FITC-conjugated secondary antibody. Cells were then fixed and labeled with rabbit anti-GFAP antibody, followed by a rhodamine-conjugated secondary antibody. CS-56 immunoreactivity was observed in a punctate pattern on the surface of the culture only after TGFβ1 treatment. Scale bar: 25 µm. (D) Astrocytes prepared from wild-type and Smad3-null mice were treated with or TGFβ1 (10 ng/ml) for 1 day and RNA was purified. Quantitative RT-PCR was performed to measure the amount of C4ST1 transcript. Upregulation of C4ST1 was not observed in Smad3-null astrocytes. (*P<0.01 compared with TGFβ1-treated WT astrocytes; ANOVA).
Movie 1. Time-lapse video microscopy shows that the growing axons from mouse DRG turn at the interface and continue to extend along the interface. CSPGs are immobilized along with Texas Red.
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