Figure 1.

Characterization of rhodopsin and its N-glycans prepared from ricin^R HEK293S cell lines. (A) HEK293S ricin^R cell lines expressing the opsin gene by transient transfection were used for purification of rhodopsin (Materials and Methods). Rhodopsin was characterized by SDS/PAGE (10%) followed by visualization of proteins by silver stain. Lanes 1 and 10 (M) (molecular weight standards); lanes 2–6, rhodopsin purified from HEK293S ricin^R cell lines obtained by using 1 ng/ml ricin; lane 7, rhodopsin purified from a ricin cell line obtained by using 10 ng/ml ricin; lane 8, rhodopsin from WT HEK293S cells; lane 9, rhodopsin from bovine ROS. (See text for further details.) (B) Rhodopsin was prepared as described above. N-glycans removed by treatment with PNGaseF were analyzed as in Materials and Methods. Profile 1, profile of molecular weight standard (malto-oligosaccharides) and the corresponding number of glucose units indicated above the profile. N-glycan profiles of rhodopsin purified from HEK293S (profile 2), one HEK293S ricin^R cell line at 1 ng/ml ricin concentration (profile 3), the HEK293S ricin^R cell line at 10 ng/ml ricin concentration (profile 4), and bovine ROS (profile 5). The profile of RNase B N-glycans is in profile 6; the peak corresponding to Man₅GlcNAc₂ is indicated by an arrow. The peaks show relative fluorescence intensity corresponding to eluting N-glycans.