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

To screen transfected clones of cells for the production of trypsinogen or Ig ~ light chain lff*-lO 5 cells were incubated for 5 h at 37°C in 15 % C02 in DME I-I21 lacking methionine and cysteine and supplemented with 2% FCS and 100-200/tCi of either [35S]methionine, 135S]cysteine, or "hans [35S]label. The culture media and cell extracts were recovered and adjusted to a final concentration of 0.3 % SDS and NDET. Samples were precleared by incubation for 30 min at room temperature with 100 pl of prewashed SAC (Pansorbin or Zysorbin). The material that nonspeeitically bound to the SAC was removed by centrifugation and the samples were incubated for 16 h at 4°C with specific antiserum, either rabbit anti-mouse ~ or rabbit antirat trypsinogen. The amounts and types of antiserum to be used for immunoprecipitations were optimized using derivatives of the mouse myeloma MPC-11 expressing K light chain and transfected wild type AtT-20 cells expressing trypsinogen (Baumal et al., 1973; Burgess et al., 1987) . Soluble immunoprecipitates were recovered by incubating with fixed SAC for 30 min at room temperature. The preparation of samples for analysis by SDS-PAGE and autoradiography were as described (Burgess et al., 1985) .