Comprehensive Western Blot Analysis Protocol

We carried out Western Blot analysis as described previously [7 (link)]. Membranes were incubated with following primary antibodies for overnight at 4 °C: SEMA5A (Invitrogen; 1:1000), anti-E-cadherin (E-cad) (HECD1; 1:100), anti-N-cadherin (N-cad) (1:100), anti-β-catenin (1:200), anti-SNAIL (Abcam; 1:500), anti-GAPDH (Santa Cruz, 1:1000) and anti-β-actin (Sigma, 1;1000). PI3K (Sigma, 1:1000), p-PI3K (p85Tyr458-p55Tyr199, Cell Signaling, 1;750), AKT (Cell Signaling 1:1000), p-AKT (Ser473, Cell Signaling, 1:1000), GSK3β (Cell Signaling, 1:1000) p-GSK3β (Ser9, Cell Signaling 1:1000). Anti-E-cad, anti-N-cad, and anti-β-catenin were generous gift from Dr. Keith Johnson, University of Nebraska Medical Center; UNMC, Omaha, NE, USA. The membrane was incubated with secondary horseradish peroxidase antibody (mouse (Sigma), 1:5000, rabbit (Thermo Scientific); 1:5000) for an hour at room temperature. We utilized NIH Image J Software for quantification of the intensity of the bands, of our protein of interest and their respective loading control. We also normalized the bands to the Control cells used in the study.