Fluorchem fc2 imaging system

Cells or tissues were incubated on ice with lysis buffer [50 mM Tris-HCl (pH 7.5), 20 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 5% glycerol, and protease inhibitors] and centrifuged at 20,000 × g at 4°C for 15 min. Supernatants were collected, and their protein concentrations were determined with a Pierce Bicinchoninic Acid Protein Assay Kit (Thermo Fisher, Waltham, MA, USA). Equal amounts of protein (30 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking for 1 h with 5% skimmed milk in Tris-buffered saline (TBS; 10 mM Tris and 150 mM NaCl), the membranes were incubated with the following primary antibodies overnight at 4°C, all of which were raised in mice and supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA): anti-CREB1 (1:1,000 dilution), anti-B-cell lymphoma 2 (BCL-2; 1:800), anti-matrix metalloproteinase9 (MMP9; 1:1,000), and anti-GAPDH (1:2,000). The membranes were subsequently washed three times with TBS and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:5,000; Santa Cruz Biotechnology) for 2 h at room temperature. Immunoreactive protein bands were detected with an enhanced chemiluminescence-based FluorChem^® FC2 imaging system (Alpha Innotech, San Jose, CA, USA).

HepG2 were seeded into six-well plates and treated with different stimulants as designated. Cells were then harvested by centrifugation and lysed using cell lysis buffer to obtain total protein. Protein concentration was determined by Bradford assay kit. Proteins (30 µg) were loaded into each lane of a 10% SDS-polyacrylamide gel and transferred onto PVDF membranes (Roche, Germany). Membranes were blocked for 1 h in 5% (w/v) nonfat milk and then incubated with primary antibody at room temperature for 1 h. After being washed three times in TBST, the membranes were incubated with a horseradish peroxidase-coupled goat anti-rabbit IgG (diluted 1:5000) for 1 h at room temperature and then washed three times. Finally, expression levels of protein were detected using an enhanced chemiluminescence system ECL (Roche). Immunoblotted bands were quantified by FluorChemFC2 imaging system (Alpha Innotech, San Leandro, CA, USA), and the protein of interest was normalized to β-actin.

Protein samples were extracted from bladder cancer cells with M-PER™(Mammalian Protein Extraction Reagent, catalog no. 78501; Thermo Fisher Scientific Inc.), Equivalent quantities of proteins (50 µg) were separated with 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk in PBS with Tween 20 and incubated at 4°C overnight with the primary antibodies, rabbit polyclonal anti-E-cadherin and mouse monoclonal anti-N-cadherin antibody, and a mouse monoclonal anti-Tubulin antibody (Cell Signaling Technology, Inc.; catalog no. T6199; dilution, 1:1,000). The cells were then incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) (anti-rabbit or anti-mouse; catalog no. 7074 and 7076, respectively; dilution, 1:1,000; Cell Signaling Technology, Inc. Danvers, MA, USA) for 1 h at room temperature. Detection of the protein bands was performed by a FluorChem^® FC2 Imaging System (Alpha Innotech, San Leandro, CA, USA).