Estrogen receptor beta

The reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA); Neutral formalin (10%), acetic acid, a bicinchoninic acid (BCA) protein assay kit, N-(1-naphthyl)-ethylenediamine dihydrochloride, sodium chloride, tetraethyl ethylene-diamine (TEMED), Trizma base, Triton X, and Tween 20.
Additional reagents and chemicals were obtained from the following manufacturers: 10% ammonium per-sulfate solution, radioimmunoprecipitation assay (RIPA) buffer, and skim milk were obtained from LPS Solution (Daejeon, Republic of Korea); bovine serum albumin (BSA) was obtained from GenDEPOT (Barker, TX, USA); 4% paraformaldehyde (PFA), 10X Tris–glycine buffer, and 10X Tris glycine-SDS buffer were obtained from XOGENE (Daejeon, Republic of Korea); protease inhibitor, phosphatase inhibitor, RNA Later, chemiluminescence (ECL) advanced kit and estrogen receptor alpha, estrogen receptor beta, and actin antibody were obtained from Thermo Fisher Scientific (Waltham, MA, USA); methylene alcohol was obtained from Daejung Chemicals & Metals Co. (Siheung, Republic of Korea); polyvinylidene fluoride (PVDF) membranes were obtained from Pall Co. (Port Washington, NY, USA).

The experimental methods for protein quantification and analysis were all adapted from our previous publication [14 (link)]. Briefly, approximately 30 mg of left ventricle tissue was excised from both groups and homogenized in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing a protease cocktail (Sigma Life Science, Burlington, MA, USA) and PMSF. The homogenized samples were centrifuged at 15,000 rpm for 30 min at 4 °C, and supernatant was quantified for 50 μg of equivalent proteins using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Each sample was denatured and loaded into an SDS-PAGE precast gel (Bio-Rad Laboratories, Hercules, CA, USA). Blots were later blocked in 5% w/v nonfat dry milk for 1 h and probed in 1:5000 dilutions of primary antibodies for Kv1.4 (AB5926) and GAPDH (Millipore) antibodies, and 1:1000 dilutions of KChIP2 (Abcam, Cambridge, MA, USA), Kv4.2 (Millipore, Billerica, MA, USA), estrogen receptor beta (Thermo Fisher, CA, USA), androgen receptor (Abcam, Cambridge, MA, USA), and Kv1.5 antibody (Santa Cruz Biotechnology, Dallas, TX, USA). The band intensities from GAPDH were used to normalize the target proteins using ImageJ software.