Chemigenius gel bio imaging system

Parental and resistant cellular pellet was obtained at 70% of confluence after three subcultures. Subsequently, protein lysates were harvested using RIPA buffer (50 mM Tris, pH 7.2; 150 mM NaCl; 1% Triton X-100; and 0.1% SDS) containing protease (1:100, Roche, USA) and phosphatase (1:100, Sigma-Aldrich, USA) inhibitors. The protein concentrations were determined using a bicinchoninic acid assay (Pierce, Thermo Scientific, USA). An amount of 40 μg of the proteins were separated by SDS-PAGE and transferred (Bio-Rad, USA) to PVDF membranes (Millipore, USA). Human SERPINA1, BTC and CCL5 protein expression levels were quantified using goat monoclonal antibody specific for human SERPINA1 and BTC (1:500, Santa Cruz Biotechnology, USA) and goat polyclonal antibody specific for CCL5 protein (1:500, R&D Systems, USA). The expression levels of these three proteins were standardized to human β-actin using a rabbit monoclonal anti- β-actin antibody (1:5000, Cell Signaling, USA). Primary antibodies were detected using donkey anti-goat or goat anti-mouse or mouse anti-rabbit- radish peroxidase (HRP)-conjugated secondary antibodies (1:5000, Santa Cruz Biotechnology, USA). Immunoreactive bands were visualized using myECL^™ Imager (Thermo Scientific, USA) according to the manufacturer’s instructions, and then quantified by densitometry using a ChemiGenius Gel Bio Imaging System (Syngene, USA).

Cells were lysed using RIPA buffer (50 mM Tris, pH 7.2; 150 mM NaCl; 1% Triton X-100; and 0.1% SDS) containing protease (1:100, Roche, USA) and phosphatase (1:100, Sigma-Aldrich, USA) inhibitors. The protein concentrations were determined using a bicinchoninic acid assay (Pierce, Thermo Scientific, USA). Sixty μg of the proteins were separated by SDS-PAGE and transferred (Bio-Rad, USA) to PVDF membranes (Millipore, USA). Protein expression levels were quantified using rabbit polyclonal antibodies specific for each protein (TGF-β, Snail and E-cadherin, Abcam, USA, PTEN and Ki67, Sigma, USA). The expression levels of these proteins were standardized to human α-actin using a mouse polyclonal anti-α-actin antibody (Millipore, USA). Primary antibodies were detected using goat anti-rabbit or goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, US A). Immunoreactive bands were visualized using Western Lighting Chemiluminescence Reagent Plus (PerkinElmer, USA) according to the manufacturer’s instructions, and then quantified by densitometry using a ChemiGenius Gel Bio Imaging System (Syngene, USA).