Ap conjugate substrate kit nbt bcip

Cytosolic and nuclear extracts were prepared using a Nuclear/Cytosol Fractionation Kit (BioVision Research, USA). Cytosolic extracts (for NF-κBp65, NF-κBp50, COX-2, iNOS, STAT3, c-Myc, Bcl-xl, Nrf2, SOD, GSTP, EGFR, Akt, p-Akt, Keap1, IKKα/β and β-actin) or nuclear extracts (for NF-κBp65, NF-κBp50, STAT3, p-STAT3, Nrf2, p-Nrf2 and lamin) were separated on 7.5%, 10% or 12% SDS–PAGE slab gels. Then, the proteins were transferred to a nitrocellulose Immobilon-P membrane. After blocking for 2 h with 10% skimmed milk, proteins were incubated with primary antibodies against NF-κBp65, NF-κBp50, COX-2, iNOS, STAT3, p-STAT3, c-Myc, Bcl-xl, Nrf2, p-Nrf2, SOD, GSTP, EGFR, Akt, p-Akt, Keap1, IKKα/β, β-actin, and lamin. Alkaline phosphatase (AP)-labeled anti-rabbit IgG, anti-goat IgG, and anti-mouse IgG secondary antibodies (Bio-Rad Laboratories, USA), as well as horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Boster Bio, USA) secondary antibodies were used in the staining reaction. Bands were visualized by the AP Conjugate Substrate Kit NBT/BCIP or the chemiluminescent HRP substrate of the Clarity ECL Kit (Bio–Rad Laboratories, USA). The amount of immunoreactive product in each lane was determined using a ChemiDoc Imaging System (Bio–Rad Laboratories, USA). Values were calculated as relative absorbance units (RQ) per mg of protein and expressed as a percentage of the control.

Cytosolic extracts for COX-2 and β-actin, or nuclear extracts for Nrf2, NF-кB p65, NF-кB p50, and lamin protein detection, were separated on 12% or 10% SDS-PAGE slab gels. β-actin and lamin were used as loading control. Proteins were transferred to the nitrocellulose Immobilon P membrane. After blocking for 2 h with 10% skimmed milk, proteins were probed with primary antibodies against Nrf2, NF-кB p65, NF-кB p50, COX-2, β-actin, and lamin. Alkaline phosphatase AP-labeled anti-rabbit IgG, anti-goat IgG, and anti-mouse IgG secondary antibodies (BioRad Laboratories, Hercules, CA, USA) were used in the staining reaction. Bands were visualized by AP Conjugate Substrate Kit NBT/BCIP (BioRad Laboratories, Hercules, CA, USA). The amount of immunoreactive products in each lane was determined using ChemiDoc Imaging System (BioRad Laboratories, Hercules, CA, USA). Values were calculated as relative absorbance units (RQ) per mg of protein and expressed as a percentage of control.

To determine the protein levels of NF-κB p50, NF-κB p65, Nrf2, COX-2, SOD, NQO1, CAT, GSK3β, p-GSK3β, Keap1, lamin, β-actin, p62 the immunoblot assays were performed. Nuclear (Nrf2, NF-κB p50, NF-κB p65, lamin) or cytosolic (Nrf2, Keap1, SOD, NQO1, CAT, p62, GSK-3β, P-GSK-3β, COX-2 and β-actin) fractions were separated on 10% and 12% SDS-PAGE gels and transferred to nitrocellulose membranes. Subsequently, skimmed milk (10%) was used as a blocking solution. After blocking, the proteins were incubated with dedicated primary antibodies. As an internal control lamin and β-actin were used. Alkaline phosphatase- and horseradish peroxidase-labeled anti-goat IgG and anti-rabbit IgG were applied as secondary antibodies. For alkaline phosphatase-labeled antibodies, bands were visualized in the staining reaction with AP Conjugate Substrate Kit (NBT/BCIP) (BioRad, Hercules, CA, USA). The products of reactions horseradish peroxidase-labeled antibodies with Clarity Western ECL Substrate (BioRad, Hercules, CA, USA) were visualized by chemiluminescence. The amount of the protein in each lane based on the intensity of the band was quantified by the Image Lab software (BioRad Laboratories, Hercules, CA, USA).

The immunoblot assay was used to determine the level of p53, Bax, and Bcl-xl proteins. Briefly, after 24 h culture with various concentrations of methoxy stilbenes or RSV, cell lysates were obtained by extracting HL-60 and THP-1 cells with RIPA buffer. The concentration of total proteins was determined using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Fifty micrograms of total protein from each concentration was resolved by electrophoresis using 12% or 10% SDS-PAGE, followed by electrotransfer onto a polyvinyl difluoride transfer membrane (Immobilon-P, Millipore). After blocking with 10% skimmed milk, the proteins were probed with anti-human p53, Bax, and Bcl-xl antibodies. The β-actin protein was used as an internal control. As the secondary antibodies in the staining reaction, alkaline phosphatase-labeled anti-goat IgG, anti-mouse IgG, or anti-rabbit IgG was used. Bands were visualized with the Bio-Rad AP Conjugate Substrate Kit NBT/BCIP. The amount of the immunoreactive product in each lane was determined using the Quantity One software (Bio-Rad Laboratories). Values were calculated as relative absorbance units (RQ) per mg protein.

Cytosolic extracts for Nrf2, SOD-1, NQO1, GSTA, COX-2, and β-actin, or nuclear extracts for Nrf2, NF-кB p65, NF-кB p50, and lamin protein detection, were separated on 12% or 10% SDS-PAGE slab gels. The β-actin and lamin were used as a loading control. Then, 100 µg of cytosolic and nuclear fractions were added per well for the SDS-PAGE. Proteins were transferred to the nitrocellulose Immobilon P membrane. After blocking for 2 h with 10% skimmed milk, proteins were probed with primary antibodies against Nrf2, NF-кB p65, NF-кB p50, SOD-1, NQO1, GSTA, COX-2, β-actin, and lamin. Alkaline phosphatase AP-labeled anti-rabbit IgG, anti-goat IgG, and anti-mouse IgG secondary antibodies (BioRad Laboratories, Hercules, CA, USA) were used in the staining reaction. Bands were visualized with the AP Conjugate Substrate Kit NBT/BCIP (BioRad Laboratories, USA). The number of immunoreactive products in each lane was determined using the ChemiDoc Imaging System (BioRad Laboratories, Hercules, CA, USA). Values were calculated as relative absorbance units (RQ) per mg of protein and expressed as a percentage of control.