Western blot analysis
Protein extracts were isolated from homogenized liver (freshly harvested and flash frozen) of wildtype, Line 525A, and Line A transgenic mice. Protein (25 μg per sample) was analyzed by electrophoresis on a 10% SDS-PAGE Ready Gel (Bio-Rad, Hercules, CA) and transferred to PVDF membrane (Sequi-Blot; Bio-Rad) via electroblotting. All blots were probed for E2 protein using polyclonal rabbit E2 antisera (1:5,000), which detects both mouse (~47 kD) and human (~54 kD) E2 subunits ([30]; gift from Dr. Susan Hutson, Wake Forest University). Blots were re-probed with a rabbit anti-c-myc tag antibody (1:10,000; abCam, Cambridge, MA; cat.# ab9106-100). Blots were also re-probed with an antibody for β-actin (43 kD; 1:10,000; abCam; cat.# ab8227-50) to allow for loading comparisons. A goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:10,000; Novus, Littleton, CO; cat.# NB730-H) was used for detection using the Western Lightning chemiluminescence reagent (Perkin Elmer, Boston, MA) and exposed to X-ray film.