Figure 8.

Juxtacrine activation primarily operates in trans. (A) CHO cells expressing either EGF-ct (left) or the EGF-hc chimera (right) were mixed with an equal number of cells expressing the EGFR and allowed to plate for 2 h. The cells were then changed to fresh medium (Con) or together with 10 μM batimastat (Bat), 10 μg/ml 225 mAb (225), or 10 μg/ml LC mAb (LC). After 18 h, the cells were extracted, and the EGFR was immunoprecipitated and analyzed for phosphotyrosine (top) or EGFR content (bottom) as described in Materials and Methods. (B) Mouse B82L cells expressing either human EGFR alone (R+) or both receptor and EGF-hcF (R+L+) were plated alone. Alternately, R+ cells were mixed together with cells expressing EGF-hcF alone (R+/L+). The number of cells on each plate was adjusted so that the net receptor number and ligand production was the same. Two hours after plating, the medium was replaced with that containing 10 μg/ml 225 mAb or 10 μM batimastat. After 18 h, the cells were processed as described in A. Numbers under the Tyr(P) bands are their relative intensities (in arbitrary densitometric units). This blot is representative of three replicated experiments.