Goat anti rabbit anti mouse secondary antibody conjugated with horseradish peroxidase

GBC-SD and SGC-996 cells (1 × 10⁷) were seeded in a cell culture dish and treated with different concentrations of UA for 48 h. A BCA assay (Shanghai, Beyotime, China) was used to determine cell protein concentrations after harvesting the treated cells and extracting proteins using RIPA buffer according to the method described by Levites et al. [25 (link)]. For western blot analysis, equal quantities (50 μg of protein per lane) of total proteins mixed with bromophenol blue (0.01%) were added in each lane and separated by SDS-PAGE, then electrophoretically transferred onto PVDF membranes. The membrane was blocked in blocking buffer (5% non-fat dry milk) for 1 h at room temperature, then incubated with anti-cleaved caspase-3, anti-cleaved caspase-9, anti-PARP, anti-Bcl-2, anti-Bax, anti-cytochrome c, and anti-β-actin antibodies in blocking buffer at 4°C overnight. This was followed by an incubation with a goat anti-rabbit/anti-mouse secondary antibody conjugated with horseradish peroxidase (1:5000; Abcam). After each incubation period, the membranes were washed three times with TBS/T. The immunoreactive bands were visualized using a chemiluminescent HRP substrate (ECL; GE Healthcare), then scanned and quantified with a Gel Doc 2000 (BioRad, USA). The results are representative of 3 independent experiments.

Cells were treated with various concentrations of baicalein (0, 15, 30, 60 and 120 μmol/l) for 48 h and then lysed in a sample buffer, followed by denaturation. The total protein concentration of the cell extracts was determined using the bicinchoninic acid assay system (Beyotime, China) with BSA as a standard. Equal quantities (80 μg protein per lane) of total proteins were separated by SDS-PAGE (8%, 12% gels) under reducing conditions. The proteins were then electrophoretically transferred to nitrocellulose membranes. The membranes were blocked with 5% skimmed milk, and incubated withanti-caspase-3, anti-Bcl-2, anti-Bax, anti-PARP, anti-p53, anti-cyclinA, anti-cyclinB1, anti-cyclinD1, anti-MMP2, anti-MMP9, anti-ZFX antibodies, respectively (1:1000; Cell Signaling Technology) at 4°C overnight. This was followed by an incubation with goat anti-rabbit/anti-mouse secondary antibody conjugated with horseradish peroxidase (1:5000; Abcam). An equal loading of each lane was evaluated by immunoblotting the same membranes with β-actin antibodies after the detachment of previous primary antibodies. Photographs were taken and the optical densities of the bands were scanned and quantified with the Gel Doc 2000 (BioRad, USA).