CHO-expressed cells. Left panel: N-glycosidase F cleavage of CHO-expressed HK4-6. Purified CHO-expressed (His)6-tagged Lewis (Lew), Fischer (Fisch), and Buffalo (Buff) were subjected to cleavage by N-glycosidase F as described in “Materials and methods.” The samples were subjected to LDS-PAGE electrophoresis and stained for protein (top, “Gel”) or subjected to the Western blot technique and tested for the presence of (His)6-tagged protein (bottom, “Blot”). Incubated in the absence of N-glycosidase F (–), in the presence of N-glycosidase F (+). The size of the protein bands (40 kDa and 37 kDa) are determined by computer analysis comparing with migration of the known standards. Right panel: tunicamycin inhibition of N-glycosylation of CHO cell–expressed (His)6-tagged Lewis rat HK. Purified CHO-expressed (His)6-tagged Lewis HK4-6 cultured in the absence (–) or presence (+) of tunicamycin were subjected to LDS-PAGE (Gel) and Western blotted (Blot) and detected for the presence of (His)6-tag as described in “Materials and methods.”
