Figure 1.
Expression and kinetics of cell surface appearance of hSlo in permanently transfected MDCK cells. (A) MDCK cells (1–2 × 106 cells), permanently transfected with c-myc-tagged hSlo, were incubated 24 h in the absence (lane 1) or presence (lane 2) of 12 mM sodium butyrate to induce hSlo expression. Subsequently, cells were metabolically labeled with 200 μCi/ml [35S]methionine-cysteine for 30 min. Immunoprecipitation of hSlo was made with anti-c-myc antibody. Proteins were resolved in SDS/PAGE and visualized by fluorography. (B) Cells previously treated with sodium butyrate were pulse-labeled for 30 min and chased for the indicated time periods. Cells then were incubated at 4°C with anti-c-myc antibody for 1 h to bind the newly synthesized hSlo that reached the cell surface. Immunoprecipitation was performed after cell lysis by using Protein A-Sepharose. The recombinant hSlo already was detected at the cell surface within the first hour of chase.