Laminin 332

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.

Different cell lines were seeded on top of glass coverslips coated with laminin-332 (Sigma). Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 5% BSA before staining. The following primary antibodies were used for immunofluorescence: FITC-conjugated α6 integrin (CD49f) (1:10, BD Biosciences), PE-conjugated β4 integrin (1:10, CD104) (BD Biosciences) and p-FAK tyr397 (1:200 dilution, Cell Signaling). To visualize p-FAK, anti-rabbit Alexa-488 (1:1000, Invitrogen) was incubated on slides for 30 minutes. F-actin was detected by staining with phalloidin conjugated to rhodamine (Invitrogen) at a dilution of 1:1000. Cells were visualised using a Zeiss Imager Z.1 microscope (Zeiss, Welwyn Garden City, UK) and representative photos were acquired using the associated software: Photoshop and Illustrator (both CS4; Adobe).

ECM proteins of interest were coated on the UV-activated sites of the coverslips in this step. A drop of either human fibronectin solution (VWR; 20 μg/ml in 100 mM NaHCO₃) or laminin-332 (Sigma-Aldrich; 10 μg/ml in 100 mM NaHCO₃) was placed on parafilm. Coverslips were then placed on top of the protein solution droplets with the coated side facing the solution and incubated for 1 h at room temperature. The resulting micropatterned ECM-coated coverslips were briefly washed in PBS and used immediately afterwards for either direct cell seeding or transferred onto the polyacrylamide gel.