Fig. 1. Newly synthesized GM130 and GRASP65 are quickly localized to the membrane. NRK cells stably expressing NAGFP were labeled with [³⁵S]-methionine/cysteine for 5 minutes and chased for 0 minutes, 3 minutes, 7 minutes, 15 minutes and 30 minutes. Cells were homogenized and post-nuclear supernatant (PNS) was recovered. PNS (S) was further separated into total membrane (M) and cytosol (C) fractions. (A) GM130, GRASP65, calnexin and NAGFP were immunoprecipitated from each fraction and analyzed by SDS-PAGE and autoradiography. The immature form (im) and the mature form (m) of NAGFP are indicated. An asterisk indicates a non-specific precipitate. The picture shown is a representative of two experiments with similar results. (B) The percentage of GM130 (closed squares), GRASP65 (closed triangles) NAGFP (open circles) and calnexin (open diamonds) recovered in each membrane was plotted for the chase time. The plots are the average of two experiments and vertical bars indicate the ranges. (C) PNS was subjected to flotation as described in Materials and Methods and separated into cytosolic (C) and membrane (M) fractions. GM130 and GRASP65 were immunoprecipitated from each fraction and analyzed by SDS-PAGE and autoradiography. The picture shown is a representative of three experiments with similar results.