To further validate our findings, both FLAG-tagged MOR N-terminal constructs were expressed in the culture medium of HEK293S GnT1- cells. These cells lack N-acetylglucosaminyl-transferase I (GnT1) activity, and consequently lack complex N-glycans. Expressed N-linked MOR peptides were purified from the culture media and separated on SDS-PAGE followed by Coomassie brilliant blue staining to visualize purified MOR peptide (Fig. 5B). The profile of N40 and D40 MOR was similar to that of the anti-FLAG immunoblotting shown in Figure 5A, when isolated from wild-type HEK293 cells (Fig. 5B, lanes 1-4). The molecular mass of MORD40 was slightly smaller compared to MORN40 (Fig. 5B, lanes 1 and 2), which is due to a difference in N-linked glycosylation (Fig. 5B, lanes 3 and 4). Moreover, when constructs were collected from the culture medium of HEK293S GnT1- cells both N40 and D40 MOR N-terminal peptide run at roughly the same molecular weight before (Fig. 5B, lanes 5 and 6) and after PNGase F treatment (Fig. 5B, lanes 7 and 8). Thus, further supporting that the differences in mobility shift observed for MOR N40 and D40 is due to differential N-linked glycosylation.
