Quantikine human egf immunoassay kit

WT and KO cells were seeded on uncoated 6-well plates and after 24 h the cells were incubated with serum-free media. After 72 h, the media was collected, and stored at −80 °C until assay. EGF in the media was assayed using a Quantikine^® Human EGF Immunoassay kit (R&D Systems), according to a procedure described by the manufacturer.

The experiments were performed using the following antibodies: Antibody against human integrin β1 subunit (P5D2) was from Developmental Studies Hybridoma Bank, University of Iowa; Mouse mAb against Smad2, rabbit mAbs against EGFR, p-EGFR, ERK1/2, p-ERK1/2, AKT, p-AKT, p-Src, and p-Smad2 were from Cell Signaling Technology; rabbit pAb to β4 integrin and goat antibody against α3 integrin were from Santa Cruz Biotechnology (Santa Cruz, CA); mouse mAbs against β1 integrin, α5 integrin, β4 integrin, αv integrin, FAK, p-FAK, and rat antibody against α6 integrin were from BD Biosciences; mAb against α-tubulin and α-SMA were from Sigma; mouse mAbs against Src was from upstate biotechnology. Alexa Fluor^® 488 and 647 goat anti-mouse IgG was obtained from Invitrogen (Life Technologies). The peroxidase-conjugate goat antibody against mouse, rabbit and goat IgG were obtained from Chemicon and Cell Signaling Technology. The TO-PRO-3 was from Molecular Probes; the selective EGFR blocker Tyrphostin AG1478, fibronectin and laminin-332 were obtained from Sigma; the Sulfo-EGS was from Thermo Scientific; and, the Quantikine Human EGF Immunoassay kit was from R&D Systems.