Recombinant human amphiregulin

Recombinant human amphiregulin was purchased from R&D Systems (Minneapolis, USA), aliquoted in sterile PBS containing 0.1% BSA, and used in final concentrations of 0.5 or 0.05 μg/ml. Neutralizing AREG antibody (clone AF262, R&D Systems, Minneapolis, USA) was used in a concentration of 1 μg/ml. Chlorhexidine (CHX) was purchased from Santa Cruz (Dallas, USA) and aliquoted in DMSO used in a final concentration of 20 μg/ml (0.002%). Control cells were vehicle-treated with the appropriate dose of DMSO. Neutralizing EGFR antibody (clone D1D4J, Cell Signaling, Frankfurt, Germany) was used in a dilution of 1 : 100. Treatment of cells was started 1 h prior to infection and was continued during the time of infection and the experiment.

Polyclonal anti-Sprouty2 antibody was obtained from Sigma. The monoclonal anti-phospho-ERK1/2 (Thr202/Tyr204) and polyclonal anti-phospho-CREB, anti-CREB, anti-ERK1/2, anti-phospho-AKT (Ser473) and anti-AKT antibodies were obtained from Cell Signaling (Danvers, MA). The polyclonal anti-COX-2 antibody was obtained from Abcam (Cambridge, MA). The monoclonal anti-α-tubulin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG were obtained from Bio-Rad Laboratories (Hercules, CA). Human chorionic gonadotropin (hCG), 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) and LY294002 were obtained from Sigma. Recombinant human amphiregulin was obtained from R&D systems (Minneapolis, MN). Wortmannin, PD98059 and U0126 were obtained from Calbiochem (San Diego, CA).