Figure 3.

FUT8 knockdown validation by GE-AAV constructs

HEK293T and FRhK-4 cells were transfected with plasmid DNA of the AAV vector plasmids containing the shRNA constructs outlined in Figure 2. (A and B) After 24 h, (A) HEK293T cells and (B) FRhK-4 were harvested and analyzed for FUT8 mRNA by real-time PCR. Samples were analyzed in triplicate. Data are presented as percentage knockdown as compared to wild-type HEK293T cells. (C) Wild-type HEK293T cells were transfected with AAV vector plasmids expressing 4L6 with or without a FUT8 shRNA construct. After 18 h, cells were washed and transferred to serum-free media for an additional 3 days. Media was aspirated and replaced with fresh serum-free media to eliminate IgG produced before full knockdown of FUT8. On day 7, supernatant was harvested and filtered to remove cell debris. IgG was purified using a protein A column and 3 μg were loaded onto 4%–12% bris-tris gels in duplicate. After transfer, one membrane was stained with anti-IgG-HRP and the other was probed with AAL-HRP lectin to visualize the presence of α1-6 fucose.