These data suggest that the effect of androgen may be via inhibition of protein degradation. 5-DHT prolongs the half-life of HERG protein degradation To confirm the aforementioned findings, we examined the Brivanib (BMS-540215) effect of androgen on HERG protein turnover rates with the cycloheximide chase assay. of the rapid component of the delayed rectifier K+ current (IKr), is largely responsible for the repolarization of action potential in cardiac myocytes (7, 8, 9). Inherited mutations or drug-induced blockade of the HERG channel can prolong QT intervals and increase the risk of lethal arrhythmia (7, 10, 11). The finding Brivanib (BMS-540215) that IKr densities in female rabbit ventricular myocytes are significantly lower than those in the male (12) indicates that the effect of androgen on ether–go-go-related gene (ERG)/IKr may contribute to the sex difference in QT intervals. Liu plasmid LRP11 antibody in pCI-Neo vector (Promega Corp., Madison, WI). The stable clones were selected by culture in medium containing G418 (800 g/ml), and confirmed by Western blot analysis and electrophysiological study. For biochemical analysis, pSG5-AR45, pSG5-AR, or green fluorescence protein (GFP) was transiently transfected into HERG-HEK293 cells with Lipofectamine2000 (Invitrogen). Briefly, HERG-HEK293 cells were cultured in RPMI 1640 medium supplemented with 3% FBS for 1 d, and then were transfected with AR45, AR, or GFP and kept in the aforementioned culture medium overnight. After serum starving for 16 h in RPMI 1640 medium with 0.5% FBS, cells were treated with androgens. For mechanism studies, inhibitors were Brivanib (BMS-540215) added to culture medium 1 h before and during androgen treatment. For electrophysiological studies, CHO cells were transiently transfected with cDNAs of HERG, AR45, and GFP by electroporation as described previously (21, 22). Briefly, CHO cells were electroporated (single 160 V pulse for 15 msec) with 4 g HERG, 4 g AR45, and 2 g GFP cDNAs in a 2-mm gap cuvette using a Gene Pulser Xcell (Bio-Rad Laboratories, Inc., Hercules, CA). After electroporation the cells were plated sparsely and grown on sterile glass coverslips in 60-mm tissue culture dishes. Cells were serum starved following the same protocol as mentioned previously in the protocol for biochemical analysis. Forty-eight to 72 h after transfection, cells were used for electrophysiological studies. Isolation of rabbit cardiac myocytes Myocytes were isolated from rabbit heart via enzymatic digestion as described previously (21, 22). The heart was quickly excised from anesthetized male rabbits (1 month), and perfused with calcium free Tyrodes solution (at 37 C) containing (in mm) 137 NaCl, 5.4 KCl, 1 MgCl2, 10 HEPES, and 10 glucose. The perfusion solution was then changed to the Tyrodes solution containing 1 mg/ml collagenase (Worthington type II) and 0.28 mg/ml protease (type XIV; Sigma-Aldrich), and perfused for 20C25 min. The ventricular tissue was then cut into small pieces filtered through a 250-m mesh screen. The cells were washed, and the calcium concentration of Tyrodes solution was adjusted to 1 1.25 mm gradually. Isolated myocytes were resuspended in M199 medium and plated into laminin (Sigma-Aldrich)-coated petri dishes. Cells were treated with androgens with the same protocols as described previously. Western blotting analysis Cells were washed with ice-cold PBS and then ice cold lysis buffer [150 mm NaCl, 25 mm Tris-HCl (pH 7.5), 5 mm EDTA, 1% Nonidet P-40, 0.4% deoxycholic acid, and EDTA-free protease inhibitor cocktail tablets (Hoffmann-La Roche Inc., Nutley, NJ)] was added. Lysates were rocked for 1 h at 4 C and Brivanib (BMS-540215) then centrifuged at 14,000 for 10 min at 4 C. The supernatants were assayed for total protein content (Bio-Rad Laboratories Protein Assay), and equal amounts (50C100 g) of cell lysate protein were subjected to SDS-PAGE analysis. Protein samples were combined with 4 SDS-PAGE sample buffer [4% (wt/vol) sodium dodecyl sulfate, 40% glycerol, 20% (vol/vol) -mercaptoethanol, 0.004% (wt/vol) bromphenol blue, and 125 mm Tris buffer (pH 6.8)] incubated for 5 min at room temperature, separated on a 8C10% SDS-PAGE, and electrophoretically transferred onto 0.2 m nitrocellulose membrane (Bio-Rad Laboratories). Membranes were blocked in 10% nonfat dry milk and 0.1% Tween 20 in Tris-buffered saline (TBS) for 1 h at room temperature, and incubated with appropriate primary antibodies [1:250 to 1 1:1000.