Figure 1.

Expression of O-acetylated gangliosides in the transfected cells. (A–D) COS-1/GD3⁺ cells were transfected with AT-1 (A and C) or pcDNAI (B and D). Sixty hours after transfection, the cells were fixed and examined by incubation with R24 (A and B) or D1.1 (C and D), followed by rhodamine-conjugated anti-mouse IgG for R24 or fluorescein isothiocyanate-conjugated anti-mouse IgM for D1.1. (E and F) HeLa/GT3⁺/AT-1 cells (E) and HeLa/GT3⁺/pcDNAI cells (F) were also fixed and examined by incubation with 493D4. (Bar = 20 μm.) (G Lower) TLC immunostaining of O-acetylated gangliosides with mAb 493D4. Gangliosides were extracted from 5 × 10⁵ HeLa/GT3⁺ (before cloning, lanes 3 and 4), HeLa/GT3⁺/pcDNAI (before cloning, lanes 5 and 6), HeLa/GT3⁺/AT-1 (before cloning, lanes 7 and 8; after cloning, cell line A in lanes 9 and 10, and cell line B in lanes 11 and 12). Lanes 1 and 2 represent 9-O-Ac-GT3 as a standard. A slower migrating ganglioside is presumably 9-O-Ac GQ3. Ganglioside samples in even numbered lanes were treated with mild alkaline hydrolysis to remove acetyl group before separation. (G Upper) Densitometric analysis of immunostained spots of each lane in the Lower panel, performed as described in ref. 13.