Protein Profiling of EMT Markers in Cell Cultures

Total cellular protein was extracted from 2D cultures as previously described [31 (link)]. Protein concentration was determined by Lowry assay and an equal concentration of protein extracts (40 µg) was loaded on SDS-PAGE gels, transferred onto a nitrocellulose membrane and immunoblotted with primary antibodies overnight. Anti-EDB-FN antibody (G4 clone) was used at 1:1000 dilution. The following primary antibodies (1:1000 dilution) were purchased from Cell Signaling Technology (Danvers, MA): anti-E-cadherin (Cat#3195), anti-Slug (Cat#9585), anti-phospho-T308-AKT (Cat#13038), anti-phospho-S473-AKT (Cat#4060), anti-pan-AKT (Cat#4691), anti-MDR1 (Cat#12683S); and anti-Histone H3 (Cat#4499) and anti-β-actin (Cat#4970) as loading controls. The anti-Phosphoepitope SR proteins (Cat#MABE50; clone 1H4) and anti-SRp40 (Cat#06-1365) antibodies were purchased from Millipore Sigma (Temecula, CA, USA) and used at 1:500 dilution. Anti-N-Cadherin antibody (Cat#76057) was purchased from Abcam (Cambridge, MA, USA) and used at 1:500 dilution. The background-adjusted pixel intensities of test proteins were normalized with those of actin controls in FIJI, and the levels were expressed as ratio of treated over non-treated cells near the respective lanes.