Horseradish peroxidase conjugate

Excised tumor tissues from xenograft implant and cells from treated and control groups were subjected to preparation of total lysate and isolation of cytosolic and nuclear fractions as described previously [34 (link), 46 (link)]. For Western blotting, 25 μg of protein was resolved over 4–20% Tris-glycine polyacrylamide gel and then transferred onto the nitrocellulose membrane. The blots were blocked using 5% non-fat dry milk and probed using appropriate primary antibodies overnight at 4°C. The membrane was then incubated with appropriate secondary antibody horseradish peroxidase conjugate (Santa Cruz Biotechnology, Santa Cruz, CA) followed by detection using chemiluminescence ECL kit (GE Healthcare Biosciences). For equal loading of proteins, the membrane was probed with appropriate loading controls. The antibodies used were anti-IKKα (Cat#2682), anti-IKKβ (Cat#2678), anti-p-IKKα/β (Cat#2697), anti-cleaved caspase-3 (Cat#9661) and anti-histone H4 (Cat#2592) from Cell Signaling Technology, Danvers, MA. Anti-NF-ĸB/p65 (sc-8008), anti-PCNA (sc-56), anti-β-Actin (sc-47778) and anti-CK18 (sc-28264) were purchased from Santa Cruz. Densitometric measurement of the bands in Western blot analysis was performed using digitalized scientific software program using Kodak 2000R imaging system.

Dorsolateral prostate tissue from treated and control groups were subjected to preparation of total tissue lysate and isolation of cytosolic and nuclear fractions as described previously [38 (link)]. For Western blotting, 25 μg of protein was resolved over 4–20% Tris-glycine polyacrylamide gel and then transferred onto the nitrocellulose membrane. The blots were blocked using 5% non-fat dry milk and probed using appropriate primary antibodies overnight at 4°C. The antibodies used were anti-IKKα (sc-7218), anti-IKKβ (sc-34673), anti-NF-κB/p65 (sc-8008), anti-NF-κB/p50 (sc-8414), anti-IκBα (sc-1643) anti-p-IκBα (sc-8404), anti-Bax (sc-493), anti-Bcl2 (sc-7382), anti-COX-2 (sc-795), anti-cyclin D1 (sc-533), anti-PCNA (sc-56), and anti-VEGF (sc-152) procured from Santa Cruz Biotechnology, Santa Cruz, CA. Antibodies including anti-cleaved caspase-3 (#9661), anti-Bcl-xL (#2762), anti-p-IKKα/β (#2078) were purchased from Cell Signaling Technology, Danvers, MA. Anti-β-actin (#A1978) was obtained from Sigma-Aldrich, St. Louis, MO. The membrane was then incubated with appropriate secondary mouse (sc-2005) and rabbit (sc-2004) antibody horseradish peroxidase conjugate (Santa Cruz Biotechnology) followed by detection using chemiluminescence ECL kit (GE Healthcare Biosciences). For equal loading of proteins, the membrane was probed with appropriate loading controls.