Figure 2.

ProEGF is cleaved in its ectodomain to release a high– molecular mass soluble 170-kD EGF into the conditioned medium. (A) MDCK cells transfected with proEGF were grown on Transwell filters and metabolically labeled with ³⁵S-Translabel for 16 h. After labeling, conditioned medium from apical and basolateral compartments was immunoprecipitated with either the ectodomain (Ecto; mAb-EGF) or cytoplasmic domain (Cyto; Ab 1417) EGF antibodies. Total cell lysates were also immunoprecipitated with these EGF antibodies. (B) Filter-grown MDCK cells expressing proEGF were metabolically labeled with ³⁵S-Translabel for 20 min. The label was then chased for 2 h. Cells were either untreated (control) or treated with 18°C temperature, 18°C→ 37°C temperature shift, monensin, or BFA. Monensin (3 μM) and BFA (15 μg/ml) were added during starvation and throughout the pulse-label and chase. For 18°C temperature experiments, cells were either chased at 18°C or placed at 18°C for 1 h and then shifted to 37°C and chased for 2 h. Total cell lysate immunoprecipitations were performed using mAb-EGF. Apical and basolateral conditioned media were collected and immunoprecipitated. Only results of conditioned medium from basolateral compartment are shown.