Anti ephx2 antibodies

Formalin-fixed, paraffin-embedded SAT sections were used for immunohistochemical analysis and immunofluorescence investigations (n = 5 each), as previously described [23 (link)]. For the immunohistochemical analysis, we used anti-GRP78 (Abcam, Cambridge, MA, USA), Imgenex anti-ATF6 (Novus, Littleton, CO, USA), and anti-EPHX2 antibodies (OriGene Technologies, Inc., Rockville, MD, USA). The immunohistochemical analysis results were quantified using ImageScope software version 11.1 (Aperio, Vista, CA, USA), as previously reported [29 (link)]. For immunofluorescent staining, tissue sections were incubated with anti-GRP78 or anti-ATF6 antibody conjugated with Alexa Fluor-488 (Bioss, Woburn, MA, USA). EPHX2 tissue sections were incubated with anti-EPHX2 antibody (OriGene Technologies, Inc., Rockville, USA) and then incubated with Alexa Fluor-488–conjugated goat anti-mouse secondary antibody (Molecular Probes, ThermoFisher, USA). Nuclei were stained using 0.05% DAPI. The sections were viewed with a Zeiss LSM 710 confocal laser-scanning microscope, and representative areas of the sections were photographed under a 40× objective.

Western Blot analysis was performed using PBMCs from lean and obese subjects (at least n = 9 for each group), differentiated 3T3-L1 cells, and 3T3-L1 preadipocytes as previously reported [43 (link)] (3 independent experiments). Briefly, whole cell extracts were prepared in RIPA buffer, and protein concentration was determined using the Bradford method using β-globulin as a standard. Protein samples (20 µg) were loaded and resolved on 12% SDS-PAGE gels. After transfer to PVDF membranes, the proteins were probed with anti-GRP78 (Abcam, Cambridge, MA, USA) and anti-EPHX2 antibodies (OriGene Technologies, Inc., Rockville, MD, USA) overnight at 4 °C. After washing, the membranes were incubated with rabbit horseradish-peroxidase-conjugated secondary antibody for 2 h at room temperature and the proteins visualized using super sensitivity West Femto-ECL reagent (Thermo Scientific, Waltham, MA, USA). Protein bands were visualized by chemiluminescence and the images captured using the Versadoc 5000 system (Bio-Rad), and the bands intensity was determined using Quantity One Software (Bio-Rad). GAPDH was used as an internal control for protein loading and was detected with an anti-GAPDH antibody (ab2302; Millipore, Temecula, CA, USA).