Constitutive and basal secretion from the endocrine cell line, AtT-20

DNA transfections of AtT-20 cells was by a calcium phosphate precipitation protocol (Moore et al., 1983b) . For stable transformation, 100 ttg (for AtT-20 F2) or 50/tg (for ART-20 WT#6) of various plasmid DNAs and 20 t~g of a selectable DNA pSV2-neo (Southern and Berg, 1982) , or pTKhygromycin resistance plasmid (pHS-53; from Dr. Dan Littman, University of California, San Francisco, CA), were mixed and applied as a calcium 1. Abbreviations used in this paper: CAT, chioramphenicol acetyltransferase; 8-Br, 8-bromide; GAG, glycosaminoglycan; PEG, polyethylene glycol; SAC, Cowan strain bacterial cells. phosphate precipitate to 10-cm dishes (",,3 × 105 cells/dish) of ART-20 cells. Stable clones were selected in either 0.25 mg/ml (for F2) or 0.5 mg/mi (for WT#6) of the antibiotic G418 (Geneticin; Gibco Laboratories, Grand Island, NY) or with 0.175 mg/ml hygromycin (Boehringer Mannheim Biochemicals). Drug-resistant clones were screened for production of the desired protein by metabolic labeling followed by immunoprecipitation of cell extracts and culture media with the appropriate antibodies and fixed protein A-containing bacterial cells. Immunoprecipitates were analyzed with SDS-PAGE followed by autoradiography. Transfected wild type ART-20 F2 cells expressing trypsinogen are as described (Burgess et al., 1987) . ART-20 WT#6 ceils expressing proinsulin were generated by Dr. Sam Green (University of California, San Francisco, CA) using the human proinsulin-containing expression vector described above.