Gtx113194

Immunohistochemistry (IHC) was performed using mouse monoclonal antibody for CYP11B2 and rat monoclonal antibody for CYP11B1 (a kind gift from Professor Celso Gomez-Sanchez22 (link)). Commercial antibodies to β-catenin (05–665, Millipore), GnRHR (ab183079, Abcam), LHCGR (GTX100008, GeneTex), and GATA4 (GTX113194, GeneTex) were also used. The sections of paraffin- embedded adrenal tumor and surrounding tissues for IHC were stained using the non-biotin-amplified method (Novolink; Novocastra Laboratories) according to the manufacturers’ protocol. Images were acquired with fluorescence microscope Olympus BX51 combined with Olympus DP72 camera and cellSens Standard 1.14 software (Olympus, Germany). The sections were quantified at 40× and 400× magnification.

Human adrenal specimens were homogenized in T-PER tissue protein extraction reagent (Thermo-Fisher) containing a protease inhibitor cocktail (Roche). A 30 μg sample of protein from each specimen was separated using SDS-PAGE and transferred onto PVDF membranes (Millipore). Visinin-like 1 (VSNL1) is upregulated in aldosterone-producing adenomas (APAs) compared with normal adrenals23 (link). CTNNB1 exon 3 mutations could be involved in APA formation due to accumulation of β-catenin and increased expression of Cyclin D124 (link). The canonical (Wnt/β-catenin-mediated) signaling functionally interacts with GATA425 (link), a marker of gonadal differentiation that is crucial in adrenal development26 (link). Therefore, the primary antibodies used were as follows: mouse monoclonal anti–active β-catenin (Anti-ABC) (05–665, Millipore), mouse monoclonal antibody for CYP11B2 (a kind gift from Professor Celso Gomez-Sanchez), rabbit monoclonal anti-Cyclin D1 (ab134175, Abcam), rabbit polyclonal anti–VSNL1 (GTX115039, GeneTex), and rabbit polyclonal anti-GATA4 (GTX113194, GeneTex). Levels of proteins were detected using chemiluminescent detection reagents (Millipore) and visualized using a UVP Biospectrum 810 imaging system (Ultra Violet Products Ltd, Cambridge, UK).