Horseradish peroxidase hrp conjugated anti mouse secondary antibody

Liver tissue was directly lysed with an extraction buffer (T-PER, Thermo Fisher Scientific Inc., Rockford, lL, USA) for 30 min on ice. After centrifugation at 13,000 g for 15min at 4°C, protein concentration in the supernatant was measured using Bradford’s reagent (Thermo Fisher Scientific Inc.). Protein (30 μg) was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a gradient gel and then transferred onto PVDF membranes. Blocking was carried out using blocking buffer [5% nonfat dairy milk in Tris-buffered saline (20 mM Tris, 150 mM NaCl, pH 7.4) containing 0.05% Tween-20] for 1 hr at room temperature. Primary antibodies were diluted 1:1,000 in a blocking buffer and incubated at 4°C overnight. The following antibodies were used: anti-LTA (mouse antibody, Invitrogen, Waltham, MA, USA). To detect antigen antibody complexes, anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, TX, USA) were diluted 1:3000 in blocking buffer and incubated at room temperature for 45 min. Immune complexes were visualized using chemiluminescent substrate (Millipore, Burlington, MA, USA) according to the manufacturer’s instructions.

Benzo(a)pyrene [B(a)P] (purity ∼98%) and curcumin (purity ∼65–70%) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Antibodies for Bax, Bcl-2, cyclin D1, β-actin, anti-mouse horseradish peroxidase (HRP) conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and caspase-3 from Abcam (Cambridge, MA, USA). Monoclonal antibody for BPDE-DNA adduct clone 5D11 was obtained from Hycult Biotechnology (Uden, Netherlands). The monoclonal antibody for proliferating cell nuclear antigen (PCNA) was procured from BD Pharmingen (San Diego, CA, USA). The anti-rabbit HRP conjugated secondary antibodies were obtained from Amersham Biosciences (Buckinghamshire, UK).