Western blotting chemiluminescence reagent

Cell mobility related and miR-429 targeting proteins, ZEB1, ZEB2, E-cadherin, and β-catenin were evaluated by western blotting, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. Briefly, 1 × 10⁶ exponentially growing T24 and TSGH8301 cells of various groups were trypsinized and washed with PBS twice. Cells were resuspended in 100 mL of radioimmunoprecipitation assay (RIPA) buffer (PIERCE, USA) contained with cocktail protease inhibitor (Thermo Scientific, USA).
Protein (30 μg) was electrophoresed for 2 hour in 8% SDS-polyacrylamide gels and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA) by electroblotter for 1 hour at 4°C with 100 voltage. Antibodies (Cell signaling, USA) raised against ZEB1, ZEB2, β-catenin, E-cadherin and GAPDH were diluted in TBST containing 5% BSA and membranes were incubated for 1 hour with gentle agitation. The blots were washed for three times with TBST and incubated with goat anti-rabbit antibody conjugated to horseradish peroxidase for 1 hour. After three successive washes with TBST, Western blotting chemiluminescence reagent (Thermo Scientific, USA) was used for protein detection.

Western blotting was used to confirm the apoptosis‐related proteins changes with different antibodies against poly ADP‐ribose polymerase (PARP), caspase‐3, and bcl‐2, and alpha‐tubulins was used as loading control (Cell signaling, USA). Briefly, 3 × 10⁵ T24 and Caki‐1 cells, and 5 × 10⁵ DU145 cells after different agent treatment were trypsinized and washed with PBS twice. Cells were resuspended in 100 μL of RIPA buffer (Thermo Scientific) contained with cocktail protease inhibitor (Thermo Scientific). Twenty μg of extracted protein was electrophoresed for 2 hours in 12% SDS‐polyacrylamide gels and then transferred to polyvinylidene difluoride membranes (PVDF) (Millipore, USA) by electroblotter with 100 voltages for 1 hour at 4°C. Antibodies were diluted in Tris‐buffered saline with 0.1% Tween 20 (TBST) containing 5% non‐fat milk and membranes were incubated for 1 hour with gentle agitation. The blots were washed for three times with TBST and incubated with goat anti‐rabbit antibody conjugated to horseradish peroxidase for 1 hour. After three successive washes with TBST, Western blotting chemiluminescence reagent (Thermo Scientific) was used for protein detection. The membrane was pictured and analyzed by BioSpectrum 810.