Readthrough of the PTC was induced/increased as full-length proteins of B4GALNT1 and its product GM2 by treatment with aminoglycosides. (A) Immunoblotting of lysates from B78 cells after transfection with B4GALNT1 cDNAs of WT, mutant M4 or M2, and cultured in the presence or absence of G418 (M4: 8 μg/ml and M2: 200 μg/ml) for 48 h after transfection. Cell lysates were applied for SDS-PAGE and then blotted onto a PVDF membrane. After blocking, the membranes were incubated with 1:500 fold-diluted anti-B4GALNT1 antibody, then incubated with HRP-conjugated anti-mouse IgG (1:2000) after washing. Protein bands on the membrane were detected by Amersham Imager 680^TM (GE Healthcare Bio-Sciences AB, Tokyo, Japan) using ImmunoStar^TM LD (Wako, Osaka, Japan) luminescence reagents. β-actin was examined to confirm equal loading of samples. (B) Flow cytometry was performed using B78 cells transfected with B4GALNT1 cDNAs of WT, mutant M4 or M2, and cultured in the presence or absence of G418 as described in (A). Cells were incubated with the primary antibody, and then stained with FITC-conjugated goat anti-mouse IgG as a secondary antibody after washing. Control cells were prepared with secondary antibody alone. Percent of positive cells in each transfectant was shown.