Horseradish peroxidase hrp conjugated anti mouse igg

Cytosolic extracts for Nrf2, NF-κBp65, NF-κBp50, SOD-1, NQO1, COX-2, and β-actin, or nuclear extracts for Nrf2, NF-κBp65, NF-κBp50, and lamin protein detection, were separated on 12% or 10% SDS-PAGE slab gels, and 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-κBp65, NF-κBp50, SOD-1, NQO1, COX-2, β-actin, and lamin. β-actin and lamin served as loading controls. Alkaline phosphatase (AP)-labeled anti-rabbit IgG, anti-goat IgG, anti-mouse IgG secondary antibodies (Bio-Rad Laboratories, USA), and horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Boster Bio, Pleasanton, CA, 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 Clarity ECL Kits (Bio-Rad Laboratories, USA). The amount of immunoreactive products in each lane was determined using Quantity One software (Bio-Rad Laboratories, USA). Values were calculated as relative absorbance units (RQ) per mg of protein.

The cell pellets were lysed in RIPA with 1 mM Phenylmethanesulfonyl fluoride (PMSF) and protease inhibitors (Sigma) to collect cellular protein; culture supernatant was precipitated by 10% TCA/acetone to collect viral protein in culture supernatant. Similar amounts of protein from each extract were subjected to SDS-PAGE analysis and were transferred to polyvinyl difluoride (PVDF) membranes (Millipore). After blocking for 1 h with blocking buffer (5% nonfat milk and 0.1% Tween-20 in PBS), the membranes were incubated with the following primary antibodies at 4 °C overnight: anti-LC3, anti-PPV, anti-cathepsin D, anti-cathepsin L and anti-β-actin. Horseradish Peroxidase (HRP)-conjugated anti-mouse IgG or anti-rabbit IgG (Boster Biological Technology Co. Ltd) were used as secondary antibodies, and ECL (Bio-Rad) was used for chemiluminescent detection according to the manufacturer’s instructions. Image J was used to analyze and quantify the intensity of the protein band.

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.