Western Blot Analysis of Protein Expression

HK1, HNE1, and CNE2 cell lines were transfected as described above. After 48 h transfection, proteins were extracted in the defined volume of RIPA lysis buffer containing 1.0 mM PMSF, protease inhibitor cocktail and DTT, and 50 μg proteins was loaded in SDS-PAGE gel electrophoresis^{10 (link)}. The proteins were transferred to PVDF Membrane (Milipore, Darmstadt, Germany) which were incubated with following primary antibodies: anti-HBP1 (Milipore, Darmstadt, Germany), anti-ZO-1, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-β-catenin, anit-MMP9, anti-NF-κB, anti-caspase 3 and cleaved caspase 3, anti-caspase 7, anti-PARP and cleaved PARP, anti-caspase 9 and cleaved caspase 9, anti-p18^INK4C (p18), anti-p21^Waft/Cip1 (p21), anti-p27^Kip1 (p27), anti-Cyclin D1, anti-Cyclin D3, anti-CDK2, anti-CDK4, anti-CDK6 (Cell Signaling Technology, Danvers MA, USA), and β-actin (ABclonal, Cambridge, MA, USA) as an internal reference. Then the PVDF Membranes were incubated with HRP-linked anti-Rabbit or anti-Mouse IgG antibody according to the isotypes of the primary antibody. The membrane was imaged using ChemiDoc MP System (Bio-Rad, Hercules, CA, USA) and the images were analyzed using Image Lab Software (Bio-Rad, CA, USA).