Reagents Cell culture media and all supplements were purchased from Invitrogen (Basel, Switzerland). All reagents for gel electrophoresis were obtained from Bio-Rad (Reinach, Switzerland). Complete EDTA-free protease inhibitor mixture tablets, PhosStop phosphatase inhibitor Ganetespib cancer mixture tablets, and NBT/BCIP ready-to-use tablets were purchased from Roche Applied Sciences (Rotkreuz, Switzerland). MEK inhibitor U0126 was obtained from Promega (D��bendorf, Switzerland). EGF and TGF�� neutralizing antibodies were purchased from R&D (Abingdon, UK). All other reagents were purchased from Sigma. Expression Vectors for AP-tagged EGFR Ligands Constructs of alkaline phosphatase (AP)-tagged EGFR ligands were kindly provided by Shigeki Higashiyama (EGF, TGF��, HB-EGF, amphiregulin, epiregulin, betacellulin) (16, 41) and Carl P.

Blobel (epigen) (17). These vectors were constructed by inserting partial cDNAs for human TGF��, EGF, amphiregulin, epiregulin, betacellulin, and HB-EGF into the 3��-end of human placental AP cDNA in a pRc/CMV-based expression vector pAIPh (16, 41). Mouse epigen was constructed by inserting a partial cDNA for mouse epigen into the 3��-end of human placental AP in the CMV-based vector APtag-5 (17). Cell Culture and Transfection of AP-tagged EGFR Ligands Cells were maintained at 37 ��C in a humified air/CO2 (19:1) environment. Human colorectal adenocarcinoma cells (Caco-2) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% (v/v) fetal bovine serum (FBS), 2 mm glutamine, 4.5 g/liter d-glucose, 100 units/ml penicillin, 100 ��g/ml streptomycin, and non-essentials amino acids (100 ��m each).

Madin-Darby canine kidney cells (MDCK) were grown in minimal essential medium (MEM) supplemented with 5% FBS, 100 units/ml penicillin, 100 ��g/ml streptomycin, and 2 mm glutamine. Transfection was performed using PEI (Chemie Brunschwig, Basel, Switzerland). 1.5 �� 105 cells per well were seeded in a 12-well plate, 24 h before transfection. Transfection mixture (100 ��l of 150 mm NaCl containing 4 ��l of PEI plus 100 ��l of 150 mm NaCl containing 1.5 ��g DNA) was incubated for 30 min at room temperature, and then added to the cells. Conditioned Medium and Meprin�� Activity Assay Caco-2 cells, grown 7 days over confluency, were cultured in serum-free medium for 16 h.

The medium was collected (referred as conditioned medium) and accumulated meprin�� was activated using trypsin (20 ��g/ml) for Anacetrapib 2 h at 37 ��C. Trypsin was inhibited using soybean trypsin inhibitor (50 ��g/ml). Active meprin�� was inhibited using actinonin (100 nm, in excess), a meprin�� inhibitor. Meprin�� activity in conditioned medium or in medium containing recombinant active meprin�� was verified using the substrate N-benzoyl-l-tyrosyl-p-aminobenzoic acid (PABA peptide) as described previously (30).