Multi analyst macintosh software for image analysis systems

Immunoblotting was performed by technicians who were blinded to the experimental groups. The protein extracts were loaded into 8-12% Bis-Tris gels in a Bio-Rad slab gel apparatus (MiniPROTEAN Tetra cell; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and electrophoretically transferred to a nitrocellulose membrane. Blots were probed with the following antibodies: EphA2 (ab5386), Ephrin A1 (ab199697), Ephrin B3 (ab101699), EphB1 (ab129103), Cadherin (ab6528), β-catenin (ab16051), AKT (ab8805), AKT1 (phospho S473) (ab18206), nuclear factor (NF)-κB p65 (ab16502), Src1 (ab5407), Src (phospho Y418) (ab4816), β-actin (ab8227) and Lamin B (ab194109) were obtained from Abcam and were used at a 1:1,000 dilution, followed by the appropriate secondary antibodies. Bands were visualised using the enhanced chemiluminescence method (Super Signal CL-HRP Substrate System; Pierce; Thermo Fisher Scientific, Inc.), scanned with a densitometer (Bio-Rad Laboratories, Inc.) and analysed quantitatively with commercial equipment (Multi-Analyst Macintosh Software for Image Analysis Systems; Bio-Rad Laboratories, Inc.). At least three independent experiments were carried out.