Neurosteroid Binding Sites on the GABAA Receptor Complex as Novel Targets for Therapeutics to Reduce Alcohol Abuse and Dependence
Figure 3
Expression of the α4, α3, and δ subunits of GABAA receptors in the hypothalamus of rats administered 56 mg/kg of DHEA for 10 consecutive days as measured by Western blot analysis. 100 μg of tissue from each area was resuspended in lysis buffer (20 mM Tris pH 8.0, 137 mM NaCl, 0.5 mM sodium orthovanadate, 2 mM okadaic acid, 10% glycerol, 1% Nonidet P40, 2% protease inhibitor) and processed for protein extraction using MicroRoto for Lysis Kit (Bio-Rad, Hercules, Calif, USA). The Bradford Method [76] was used to determine protein concentration, and then samples were diluted, separated by SDS-PAGE, and transferred to nitrocellulose PDVF membranes (Amersham Biosciences, Piscataway, NJ, USA). The membranes were immunoblotted for two hours at room temperature with two specific antibodies, a rabbit anti-α4 antibody at a 1 : 500 dilution (Santa Cruz Biotechnology, Santa Cruz, Calif, USA), and a mouse anti-β-actin diluted in a proportion of 1 : 2000 (Santa Cruz Biotechnology). A specific secondary antibody (PerkinElmer Life Sciences, Waltham, Mass, USA) followed at a dilution of 1 : 2000. Expression was visualized using ECL Plus (PerkinElmer) and a Fuji Film luminescent image analyzer (LAS-1000 Plus, Fuji Photo Film Co. Ltd., Tokyo, Japan). The images were then quantified by densitometry using the Image Gauge program [77], and the expression value of each subunit was normalized to β-actin values.
