It has been shown that the A112G substitution in mouse MOR causes a deficit in N-glycosylation 19–21, which might underlie the differential sensitivities of MOR modulation on synaptic transmission. However, this has not been shown in human N40D MORs. To address this, we used HEK293 cells to express FLAG-tagged N-terminal (amino acids 1-67) domain of human MORs. Media was collected ~ 5 days post transfection from FLAG-MOR (1-67) N40 or FLAG-MOR (1-67) D40 containing HEK cells and subjected to SDS-PAGE and Western Analysis (Fig. 5). Anti-FLAG immunostaining revealed the molecular mass of recombinant MORN40 was higher than that in MORD40 cell lysates (Fig. 5A, left side lane 1 compared to lane 2). We next tested whether the differences observed in the masses of human MOR in N40 and D40 are due to variations in N-linked glycosylation from the predicted loss of one putative glycosylation site (Asn40) in D40 MOR peptide. To this end, we treated cellular lysates with PNGase F, an enzyme which removes all N-linked glycans. Western blotting of FLAG-MOR using an anti-FLAG antibody showed that MOR N40 and
