Western Blotting of Hep-2 Cell Lysates

Western blotting was performed as previous reported [36 (link), 37 (link)]. Briefly, Hep-2 cells were grown in the mid-log phase, washed with phosphate buffered saline and lysed for 30 min on ice. The lysis buffer with pH 6.8 contained 10% glycerol, 5% 2-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), 62.5 mmol/L Tris-HCl. The SDS–PAGE analysis was performed and membranes were incubated overnight at 4°C with antibodies directed against the indicated proteins. The antibodies were obtained from the following companies: anti-TrkB, anti-β-actin, anti-p-Akt, anti-cyclin D1, anti-p-s-Src and anti-c-Src were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-RUNX3 and anti-Keap1 were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-Twist-1 and anti-Twist-2 were purchased from BD Biosciences (San Jose, CA, USA). Membranes were washed, incubated with the appropriate secondary antibodies and exposed with the ECL chemiluminescent substrate kit (Pierce, Rockford, IL, USA). Images were analyzed with ImagePro (Media Cybernetics, Bethesda, MD, USA) and densitometry data were analyzed by using either conventional Student’s t-test. Results are reported as mean ± SD. A P-value <0.05 was considered significant and all were two-tailed.