Y397 fak

MDA-MB 231 and SUM159 cells were grown in 10 cm dishes, exposed to the treatments and then lysed as previously described [48 (link)]. Equal amounts of whole protein extract were electrophoresed through a reducing SDS/8 and 10% (w/n) polyacrylamide gels, electroblotted onto a nitrocellulose membrane (Amersham Biosciences, GE Healthcare, Milan, Italy), and probed with primary antibodies against Y397-FAK (Cell Signaling Technology, Milan, Italy), FAK (H-1) (Santa Cruz Biotechnology, DBA, Milan, Italy), phosphorylated ERK1/2 (E-4) (Santa Cruz Biotechnology, DBA, Milan, Italy), ERK2 (C-14) (Santa Cruz Biotechnology, DBA, Milan, Italy), p-AKT1/2/3 (Ser 473)-R (Santa Cruz Biotechnology, DBA, Milan, Italy), AKT/1/2/3 (H-136) (Santa Cruz Biotechnology, DBA, Milan, Italy), c-FOS (H-125) (Santa Cruz Biotechnology, DBA, Milan, Italy), EGR1 (C19) (Santa Cruz Biotechnology, DBA, Milan, Italy), CTGF (Origene, DBA, Milan, Italy) and β-actin (C2) (Santa Cruz Biotechnology, DBA, Milan, Italy). Proteins were detected by horseradish peroxidase-linked secondary antibodies (Santa Cruz Biotechnology, DBA, Milan, Italy) and then revealed using the ECL™ Western Blotting Analysis System (GE Healthcare, Milan, Italy).

5 × 10⁴ MDA-MB 231 and SUM159 cells were grown on 6 well plates. When reached 50% confluence, cells were serum-deprived for 12 h and then treated for 30 min with IGF-1. Next, cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.2% Triton X-100, washed three times with PBS and incubated overnight with or without (negative control) a rabbit primary antibody against Y397-FAK (Cell Signaling Technology, Milan, Italy). After incubation, the wells were extensively washed with PBS and incubated with donkey anti-rabbit IgG-FITC (1:400; purchased from Alexa Fluor, Life Technologies, Milan, Italy) for 1 h at room temperature. Finally, cells were washed with PBS and incubated in PBS buffer containing 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (1:1000; Sigma-Aldrich, Milan, Italy) 10 min at room temperature for nuclear staining. Images showing focal adhesion points among the cells were acquired on the Cytation 3 Cell Imaging Multimode Reader (BioTek, Winooski, VT, USA) and analyzed using the software Gen5 (BioTek). The same procedure was also applied for FAK (Cell Signaling Technology, Milan, Italy) staining in MDA-MB 231 PTK2 WT and KO as well as in SUM159 PTK2 WT and KO. A total of 10 images for each condition was detected on the Cytation 3 Cell Imaging Multimode Reader (BioTek) and analyzed using the software Gen5 (BioTek).