Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network

GH4C1 cells, obtained from L . Roman (University of Texas, Dallas, TX), were grown in a 1:1 (vol/vol) mixture of DMEM and Ham's F10 medium supplemented with 15% heat-inactivated (1 h at 55°C) horse serum, at 37°C in 5 % C02 in air. Cells were routinely passaged once a week and received fresh medium 3 and 5 d after plating. lb stimulate the accumulation of secretory granules, GH4C1 cells received 10 nM epidermal growth factor (Collaborative Research, Bedford, MA), 1 nM 170-estradiol (Sigma Chemical Co ., St . Louis, MO) and 300 nM insulin (Sigma Chemical Co.) for 48 h before radiolabeling or immunofluorescence analysis which was performed on day 7 after plating . lb analyze the effect of these hormones on the rate of synthesis of Sgll and prolactin, control and hormone-treated cells were preincubated for 30 min in methionine-free DMEM/Ham's F10 and then labeled for 30 min with fresh methionine-free medium containing 100 14Ci/ml [35 S]methionine . After three washes with calcium-and magnesium-free PBS, GH4C1 cells were solubilized in 10 mM Tris, pH 7.6, 1%