Anti zfx

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).

At 72 h post-transfection, cellular proteins were extracted from HeLa cells with RIPA buffer (Boston BioProducts, Boston, MA, USA) containing 5 mM sodium fluoride (NaF), 1 mM sodium vanadate (Na₃VO₄), and 1 mM phenylmethylsulfonyl fluoride (PMSF), supplemented with 1% Protease Inhibitor Cocktail (100X, Thermo Scientific, Rockford, IL, USA). Protein concentration was measured with the Bradford Assay. 50 µg of protein was loaded and separated in an 8% polyacrylamide SDS-gel and electrotransferred onto polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies, followed by incubation with matching HRP-conjugated secondary antibodies. Color development was carried out using the ECL reagents (Pierce, Rockford, IL, USA). GAPDH was detected and the results were used as protein loading controls. The primary antibodies used were: anti-ZFX (1:1000, Cell Signaling, Beverly, MA, USA) and anti-GAPDH (1:1000, Santa Cruz, TX, USA).