Nuclear extraction kit

Electrophoretic mobility shift assay (EMSA) was conducted to evaluate the DNA-binding activity of STAT-3 in genipin-treated HCC cells. In brief, following transfection of HCC cells for 72 h, nuclear proteins from each sample were extracted with a Nuclear Extraction kit (Sigma, USA) and subjected to EMSA following the manufacturer’s standard protocol using the LightShift® Chemiluminescent EMSA kit (Thermo Fisher Scientific, USA). The STAT-3 target probe was synthesized with a 3′-biotin modification (Invitrogen, USA) and the sequence was 5′-ACG AAC CAT TACGCTCGA CAG CCG-3′, in which the binding region is underlined. EMSA was conducted with STAT-3 EMSA Kit (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. STAT-3 oligonucleotides with infrared dye-labels were as follows: 5′-CTACGGACGTACGAACTGCACGGC-3′ and 3′-ACCTGGACTAACGTCAGCCGCG-5′.

Cell and tissue lysate homogenates were prepared as previously described [25 (link)]. Nuclear extracts were prepared using a nuclear extraction kit (Sigma-Aldrich, MO, USA). After quantification with the BCA Protein Assay Kit, equal amounts of protein samples (40 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore, MA, USA). After being blocked in TBST buffer (50 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.6) containing 5% skim milk for 2 h at room temperature, the membranes were incubated with appropriate primary antibodies against SIRT1 (1 : 1000), Nrf2 (1 : 1000), NQO-1 (1 : 1000), HO-1 (1 : 1000), gp91^phox (1 : 500), Bax (1 : 1000), Bcl-2 (1 : 1000), cytosolic cytochrome c (1 : 1000), cleaved caspase 3 (1 : 1000), histone H3 (1 : 1000), and β-actin (1 : 1000) overnight at 4°C. Then membranes were washed in TBST and reacted with a secondary horseradish peroxidase-conjugated antibody (1 : 5000) for 1.5 h at 37°C. Antigen-antibody complexes were then visualized using enhanced chemiluminescence reagents. The density of the immunoreactive bands was analyzed using Image Lab software (Bio-Rad Laboratories, CA, USA).

For the isolation of cytoplasmic and nuclear fractions from A549 cell cultures, NuCLEAR Extraction Kit (Sigma-Aldrich Sweden AB, Stockholm, Sweden) was employed following the manufacturer’s instructions. Briefly, cells from one 10-cm petri dish per condition were centrifuged, and cell pellets were incubated with 300 μL hypotonic lysis buffer (cytoplasmic fraction). Nuclei were resuspended in 100 μL of nuclear extraction buffer. For immunoblotting, 30 μL of the cytoplasmic fraction and 10 μL of the nuclear were subjected to electroforesis in either 10% or 15% polyacrylamide gels. Lamin B1 and tubulin were used as markers for nuclear and cytoplasmic compartments, respectively.