Anti mouse igg secondary antibodies

DSF and copper chloride (Cu) were purchased from Sigma-Aldrich (St Louis, MO, USA). Phosphatase inhibitors and protease inhibitor cocktail tablets were purchased from Roche Applied Sciences (Penzberg, GER). The following primary antibodies were used: p-JNK, JNK, p-cJun, cJun, p-p38, phospho-Akt, and total-Akt (Cell Signaling, Beverly, CA, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-mouse IgG secondary antibodies were purchase from Bio-Rad Laboratories (Hercules, CA, USA).

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.