Figure 3.

(a) Effect of enzymatic (heparatinase III; Hep III; or chondroitinase ABC; Chase ABC) removal or desulfation (NaClO₃ treatment) of cell-surface HSs and CSs on FGF-2-induced proliferation in SK-LMS-1 cells. Anti-HS mAb 10E4 and anti-CS mAb CS56 were used to verify the extent of cell surface removal of the corresponding GAGs. In “Rescuing” experiments, siRNA-treated cells were exposed to the glycanated (sNG2-CS⁺) or non-glycanated (sNG2/CS⁻) recombinant ectodomains of rodent NG2 (Inset; p<0.001 by Mann-Whitney U Test), or were transfected with a wild type (rat NG2) or modified rodent NG2 construct in which the primary GAG-attachment site was mutated (rat NG2^GAG; p<0.0001 by ANOVA). Dashed t₀ line indicates the starting amount of cells/well. (b) Comparative levels of FGF-2-induced mitosis observed in SK-LMS-1 cells overexpressing full-length NG2 (NG2⁺⁺; asterisk, p<0.001 by ANOVA), the NG2 ectodomain (NG2^extra mutants), or a transmembrane-cytoplasmic segment (NG2^cyto mutants; see Materials and Methods). (c) Phosphorylation pattern of the ERK-dependent Thr²³¹⁴ residue of the cytoplasmic domain of NG2 upon stimulation of the cells with the indicated growth factors (IGF-1 was used as a control), as determined by immunoblotting using antibodies to the xTpP phosphorylation motif.