Fig. 1. Evaluation of chondroitin sulfate content by immunoblotting. Immunoblot analysis with rabbit anti-NG2 antibody was performed on detergent extracts of B28 cells transfected with wild-type and mutant NG2. Half of each sample was treated with chondroitinase ABC to remove chondroitin sulfate chains (+), while the other half was left untreated (-). In the case of B28/NG2.6 cells, which express wild-type NG2 (c6/wt), this chondroitinase treatment converts the high-molecular-weight smear representing the mature proteoglycan (asterisk) into the 300 kDa core glycoprotein (arrowhead). A similar increase in the quantity of the core is seen with extracts of cells expressing the NG2/S1342A mutant (clones 42 and 44), indicating that this form of NG2 still contains chondroitin sulfate. By contrast, chondroitinase treatment has no apparent effect on extracts of cells expressing the NG2/S999A mutant (clones 51 and 65), suggesting that this form of NG2 is not modified with chondroitin sulfate.
