Fig. 5. Depletion of C4ST1 in TGFβ1-treated astrocytes reduces the production of 4-sulfated GAG chains as well as repellent activity of conditioned medium. (A) Conditioned medium derived from C4ST1 siRNA-transfected astrocytes treated with or without TGFβ1 was subjected to ELISA to measure the amount of 4-sulfated GAG chains with MAB2030. (B) C4ST1 protein level in the cell lysates was checked by immunoblotting with anti-C4ST1 peptide antibody. Actin was used as loading control. (C) Disaccharide composition analyses of conditioned medium (CM). The values obtained from three experiments were used to calculate pmol of HexUA-GalNAc(4-O-sulfate) (Di-4S) per 1 ml conditioned medium. Data are expressed as the mean ± s.d. (D) Axonal guidance spot assay. More axons that crossed the interface were observed in C4ST1 siRNA conditioned medium compared with TGFβ1 conditioned medium (^*P<0.01 compared with scrambled siRNA without TGFβ1 and C4ST1 siRNA with TGFβ1; ANOVA).