Alkaline phosphatase linked secondary antibody

After boiling in Laemmli’s sample buffer, proteins were resolved on 7% or 12% SDS-polyacrylamide gels, and transferred to the PVDF membrane. Membranes were incubated with a primary antibody. GR was detected using an anti-GR antibody (PA1 511A) (Affinity BioReagents, Golden, CO, USA). SOD1, SOD2, CAT, GSH-Red, GSH-Px, GLUT2, and 11βHSD1 were detected using Abcam (Abcam, Cambridge, UK) antibodies (ab13498, ab13533, ab16731, ab16801, ab22604, ab54460, and ab393364, respectively). β-actin was detected by AC-15 antibody (Sigma-Aldrich, St. Louis, MO, USA). NF-κB-p65 subunit, lipin-1, and lamin B were detected using antibodies from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Dallas, TX, USA) (sc-372, sc-98450, and sc-6217, respectively). After being incubated with alkaline phosphatase linked secondary antibody (Amersham Pharmacia Biotech, Little Chalfont, UK), immunoreactive proteins were visualized by the enhanced chemifluorescence method (Amersham Biosciences). Quantitative analysis of immunoreactive bands was done using ImageQuant software. β-actin was used as equal load controls for whole cell extracts and cytosols, and lamin B for nucleosols.

Cultured cells were lysed in a cell-lysis buffer (10 mm Tris-HCl (pH 7.4), 1% SDS, 1 mm Na3VO4, and a cocktail of proteinase inhibitors). The protein concentration was determined using Nano Drop 2000 (Thermo Scientific, Waltham, MA, USA). The protein extracts were subjected to Western blotting using the primary antibodies described above. The antibody-reactive protein bands were visualized using an alkaline-phosphatase-linked secondary antibody and an enhanced chemifluorescence system (Amersham Biosciences, Amersham, UK). The relative abundance of the proteins was semi-quantified by scanning the bands with the NIH ImageJ software and expressed as a ratio against the identically quantified β-actin control. The results represented at least three independent experiments.