Pulse-chase experiments of wildtype and A118G mutant of hMOPR (A118/N40-hMOPR and G118/D40-hMOPR, respectively) stably expressed in CHO cellsCells were metabolically labeled with [35S]Met/Cys at 37°C for 60 min (pulse). Medium was aspirated, and cells were incubated with complete medium (chase) for specified time periods. Cells were solubilized and immunoprecipitated with HA.11 antibody twice in tandem. Immunoprecipitated materials were resolved with SDS-PAGE and gels were dried. The gels were then exposed to a storage phosphor screen for 5 days, and the autoradiograms were acquired. (A) shows a representative autoradiogram. The intensities of [35S]-labeled protein bands were analyzed and quantified for the mature forms (B) and the precursors (C) as described in Methods. The signals for mature forms and precursors were normalized against their maximal signals observed at time points 2h and 0h, respectively. The experiment was performed three times with similar results. Each value in (B) and (C) is mean ± s.e.m. (n=3).
