Easyblot anti rabbit igg hrp

The brain tissues were collected and then homogenized on ice using T-PER (Thermo Scientific, Waltham, MA, USA) buffer with protease inhibitor cocktail set III (Calbiochem, Merck, Germany) and phosphatase inhibitors (Cayman Chemical, Ann Arbor, MI, USA). Protein concentrations were determined using the Bradford reaction. Aliquots (20 μg) were solubilized in a Laemmli buffer with 2 % mercaptoethanol (BioRad, Hercules, CA, USA) and subjected to 10 % SDS-polyacrylamide gel electrophoresis as described previously (Gdula-Argasińska et al. 2015 (link)). We used primary antibodies: anti-COX-2 (diluted 1:500), anti-cPGES (diluted 1:1000), anti-Nrf2 (diluted 1:100), anti-AhR (diluted 1:500), and anti-GAPDH (diluted 1:1000) (GeneTex Inc., Irvine, CA, USA), as well as NF-ĸB p50, NF-ĸB p65 (Cayman Chemical), diluted 1:100 and anti-GLUT4 (Sigma-Aldrich, Saint Louis, MO, USA), diluted 1:200 in Signal + for Western Blot (GeneTex). The secondary antibody was EasyBlot anti-rabbit IgG (HRP) diluted 1:1000 in Signal + for Western Blot (GeneTex). Proteins were detected using a Clarity Western ECL luminol Substrate Western blotting detection kit (Bio-Rad). The integrated optical density of the bands was quantified using a ChemiDoc Camera with Image Lab software (Bio-Rad).