N-linked glycosylation and Western analysis. Lysates (A) and culture medium (B) from Huh7 and CHO-K1 cells transiently transfected with plasmid constructs expressing WT or A267T PC were harvested 48 h after transfection and treated ± Endo-H for 18 h at 37°C. After completion of the reaction, the samples were analyzed by 10% SDS-PAGE under reducing conditions. Following blotting onto a PVDF membrane and subsequent incubation with antibody against PC, the bands were visualized with ECL. Equal amounts of PC (as measured by ELISA) in cell lysates and culture medium were analyzed.