Western blot analysis of NFKB pathway activation

SDS-PAGE was performed with pre-cast stain-free 4–20% gels (Mini-Protean TGX, Bio-Rad). Precision Plus Protein All Blue Prestained Protein Standards were used for size determination of proteins (Bio-Rad). After transfer to polyvinylidene difluoride (PVDF) membranes by electroblotting (Transblot® Turbo, Bio-Rad), membranes were incubated in blocking solution (5% non-fat dry milk (Bio-Rad) in PBS containing 0.05% Tween, PBS-T), followed by a primary rabbit anti-p65 antibody (0.5 μg/ml, Abcam, diluted in 5% non-fat dry milk in PBS-T). Swine anti-rabbit IgG horseradish peroxidase (HRP, Dako, Glostrup, Denmark), diluted 1:1700 in 1% non-fat dry milk in PBS-T, was used as secondary antibody. Signals from HRP-conjugates were detected using Clarity Western ECL Substrate (Bio-Rad). Re-blotting against β-actin (cytosolic fraction) or Lamin B1 (nuclear fraction) were performed by using a primary mouse anti-actin antibody (Abcam, diluted 1:10,000 in 1% non-fat dry milk in PBS-T) or rabbit anti-Lamin B1 antibody (Abcam, diluted 1:10,000 in 5% non-fat dry milk in PBS-T). Goat anti-mouse IgG-Alexa Fluor 488 (Invitrogen, diluted 1:5000 in 1% non-fat dry milk in PBS-T) or swine anti-rabbit IgG-HRP (Dako, diluted 1:1700 in 1% non-fat dry milk in PBS-T) were used as secondary antibodies. Signals from HRP-conjugates were detected using Clarity Western ECL Substrate (Bio-Rad). Membranes and gels were imaged and analyzed using the ChemiDoc™ MP System (Bio-Rad).