L is synthesized in differently modified forms due to N-glycosylation. (A) COS-7 cells and HEK 293T cells were either transfected with empty control plasmids (control, C) or transfected with vectors carrying the cloned L gene (L). HuH-7 cells were transfected with a cloned replication-competent HBV genome (HBV). Two days after transfection, microsomal vesicles were prepared and analyzed by SDS-PAGE and L-specific immunoblotting with the preS1-specific monoclonal antibody MA18/7. (B) The EcR-LHA-293 cell line with inducible L expression was either mock-treated (control, C) or induced with PonA. Cell lysates were mock-digested or digested with PNGase F, tunicamycin, or Endo H, as denoted above each panel. Samples were resolved by SDS-PAGE and subjected to L-specific immunoblotting. The nonglycosylated p39 and single-glycosylated gp42 forms of L are indicated on the right of each panel, and arrows to the right point at the two novel forms of L.