Fig. 2. Newly synthesized GM130 and GRASP65 are localized to Golgi membranes. (A) NRK cells stably expressing NAGFP were labeled with [35S]-methionine/cysteine for 5 minutes and homogenized immediately (upper gallery) or after 30 minutes chase (lower gallery). Post-nuclear supernatant was obtained and analyzed by Nycodenz density gradient as described in Materials and Methods. To the left of the panels (lane 1) is the top (lightest) fraction and the right (lane 10) is the bottom (heaviest) fraction. GM130, GRASP65, calnexin and NAGFP were immunoprecipitated from each fraction and precipitated materials were analyzed by SDS-PAGE and autoradiography (indicated by arrows). Asterisks indicate nonspecific precipitates. The picture shown is a representative of three experiments with similar results. (B) Bands in (A) were quantified by densitometry and percentages of the total sum of the precipitated material in all fractions were calculated and plotted for each fraction.