Fig. 3.

TNF-α converting enzyme (TACE) mediates TNF transactivation of ErbB4 in colonic epithelial cells. YAMC-ErbB4 cells were incubated for 30 min with 50 μM of the broad spectrum metalloproteinase inhibitor GM6001 (A) or 10 μM of the TACE selective inhibitor TNF-α protease inhibitor (TAPI)-1 (B), then stimulated with TNF (100 ng/ml, 30 min) or HRG (1 ng/ml, 10 min). C: YAMC-ErbB4 cells were treated with 100 ng/ml TNF for various time points or with 20 ng/ml PMA for 1 min. Cell lysates were analyzed by Western blot with antibodies specific for phosphorylated TACE (PT-735), ERK, or total actin. D: TACE^−/− colonic epithelial cells infected with ErbB4 and with either wild-type TACE or vector added back were treated with 100 ng/ml TNF for 30 min or 10 ng/ml HRG for 10 min. E: YAMC-ErbB4 cells were treated directly with 100 ng/ml TNF for 5 or 15 min or 10 ng/ml HRG for 10 min. TACE^−/− colon epithelial cells expressing ErbB4 were treated with conditioned media (CM) from the corresponding YAMC cells for 10 min. Cell lysates were then analyzed by Western blot with antibodies specific for phosphorylated ErbB4 (PY-1284), total ErbB4, or actin. All blots are representative of at least 3 experiments.