(c) Testosterone EIA
Plasma samples were thawed on ice and 20 µl of plasma from each individual was aliquoted into glass tubes. Hormone was extracted using diethyl-ether mixed by agitating and incubation at room temperature for 20 minutes. After incubation, all tubes were snap frozen and supernatant was immediately transferred to a fresh tube. This procedure was repeated three times to extract the hormone and finally the tube was dried using high pressure nitrogen gas. 250 µl of assay buffer was added to each tube and 100 µl of extracted hormone was used in duplicate wells to run testosterone assay. Plasma testosterone was measured using a high sensitivity testosterone ELISA kit (Enzo ADI-900-176) as per manufacture’s protocol. All samples were run on one assay plate with an intra-assay coefficient variability of 0.6% ± 0.06 (mean ± SE) and sensitivity of 2.6 pg/ml.
Measurement of protein expression
Lysate preparation and protein digestion
Individual tissue samples were ground in a Dounce homogenizer (Kimble, catalog no 885301) in 8 M urea (Catalog no 8648-01, Mallinckrodt) in 100 mM ammonium bicarbonate (Catalog no 3003-10, JT Baker) with 20 passes. Lysed proteomes were then subjected to a low-speed spin (1400 x g, 5 min) to remove debris, and ultracentrifugation (100,000 x g, 45 min) to separate membrane and cytosolic fractions. The supernatant was removed and saved as the soluble proteome, while the pellet was washed and resuspended in cold PBS by sonication or in isotonic resuspension solution (20 mM Hepes, 2 mM DTT) by pipetting and saved as the membrane proteome. Total protein concentration for each proteome was determined using a Bio-Rad Dc Protein Assay kit, and proteomes were kept at -80°C until further use. Samples were resuspended and denatured in 8M urea with 100 mM ammonium bicarbonate, p H 7.8. Disulfide bonds were reduced by incubation for 45 min at 57 °C with a final concentration of 10 mM Tris (2-carboxyethyl) phosphine hydrochloride (Catalog no C4706, Sigma Aldrich). A final concentration of 20 mM iodoacetamide (catalog no. I6125, Sigma Aldrich) was then added to alkylate these side chains and the reaction was allowed to proceed for one hour in the dark at 21 °C. Aliquots of 100 μg protein were taken and diluted to 1 M urea using 100 mM ammonium bicarbonate, p H 7.8. Two μg of trypsin (V5113, Promega) was added and the samples were digested for 14 hours at 37 °C.