Figure 1.

Generation of a FUT8 KO cell line

HEK293T cells were transfected with three gRNA targeting FUT8 and a CAS9/GFP expression plasmid. (A) After 24 h, cells were sorted and the 20% highest GFP expressing cells were individually sorted into a 96-well plate and cultured until confluent. (B) Four of the FUT8 KO clones isolated from cell sorting and a HEK293T wild-type control were transfected with a 10-1074 expression plasmid. After an additional 4 days, supernatant was harvested and filtered to remove cell debris. IgG was purified by protein A column and 3 μg were run on 4%–12% bris-tris gels in duplicate. After the 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.