Samples were loaded on 7.5% (EGFR) or 10% (ERK1/2) SDS-polyacrylamide gels and subsequently electroblotted onto a polyvinylidene difluoride (PVDF) membrane (Hybond P, Amersham Biosciences). Blocking was done overnight at 4 ��C using T-TBS (25 mm Tris-HCl pH 7, 150 mm sodium chloride, 2.5 mm potassium chloride, 0.1% Tween-20) containing 5% dry milk. The membrane most was then incubated with the first antibody (pEGFR 1:5000, pERK1/2 1:200, EGFR 1:10000, ERK1/2 1:200) in T-TBS containing 2% dry milk for 1 h at room temperature and with the appropriate horseradish peroxidase-conjugated secondary antibody (1:25,000) for 1 h at room temperature. Immune complexes were visualized using enhanced chemiluminescence (ECL Plus, Amersham Biosciences) on x-ray films. The membrane was stripped using 62.

5 mm Tris-HCl pH 6.2, 2% SDS, 50 mm DTT on a shaking plate at 65 ��C for 30 min followed by washing steps in T-TBS. Densitometric analysis of Western blots were performed using Image J software (Wayne Rasband, NIH). Cell Treatment for Proliferation and Migration Experiments For proliferation and migration experiments Caco-2 cells were stimulated with conditioned medium containing activated meprin��, inhibited meprin��, or 100 ng/ml EGF (positive control). Inhibitors were added to media containing active meprin�� at the beginning of the treatment (2 ��g/ml neutralizing EGF and TGF�� antibodies, 10 ��m EGFR inhibitor AG1478, or 10 ��m MEK inhibitor U0126). Cells were pretreated with neutralizing antibodies and EGFR inhibitor for 2 h.

Alamar Blue Cell Proliferation Assay Alamar Blue uses the natural reducing power of living cells to convert resazurin to the fluorescent molecule, resorufin (42). Caco-2 cells were seeded at a density of 1000 cells per well in a 96-well plate. After 48 h of incubation, cells were washed twice using phenol red-free medium, followed by stimulation for 24 h as described above. For the last 3 h, Alamar Blue (43) solution was added to a final concentration of 10 ��g/ml. Fluorescence at 595 nm was measured directly (0 h) and 3 h after addition in a multilabel plate reader (2300 EnSpire multilabel reader; Perkin-Elmer, Turku, Finland). Values obtained at time point 0 h were subtracted from those obtained at time point 3 h (20 replicates/condition, n = 3 experiments). Cell Titer Glo Cell Viability Assay This assay is a method to determine the number of viable cells in culture based on quantification of the ATP present.

Consequently, ATP levels represent the number of metabolically active cells (44). 1000 Caco-2 cells/well were seeded in a 96-well plate. After 48 h, cells were serum-starved Drug_discovery overnight followed by 24 h treatment with the different stimuli. Subsequently, 100 ��l of Cell Titer Glo reagent were added to each well, cells were incubated for 10 min in the dark, and luminescence was measured in a 2300 EnSpire multilabel plate reader (3 replicates/condition, n = 3 experiments).