Fig. 5. A GFP TA construct tagged with a lumenal N-glycosylation consensus sequence acquires an N-linked oligosaccharide that undergoes processing in the Golgi complex. Lysates prepared from cells expressing GFP-22-Nglyc (lanes 1, 2, 4, 5) or from non-transfected MDCK cells (lane 3) were either mock-digested (lanes 1, 3, 4) or digested with PNGase F or Endo H, as indicated, and then analysed by SDS-PAGE followed by western blotting with antiopsin antibodies. The glycosylated forms of the construct are both converted by PNGase F to a more rapidly migrating form, whereas the major, higher M_r band is resistant to Endo H. The vertical arrow in lanes 1 and 4 indicates the Endo-H-sensitive polypeptide, presumably localised in the ER. Numbers on the right indicate the position and size (kDa) of markers (Bio-Rad).