Trypsin

Protein samples were run through strong cation exchange (SCX) liquid chromatography in order to fractionate samples of major proteins. Samples were reduced by addition of reducing agent (10 mM DTT, Sigma) in 50 mM ammonium bicarbonate (ABC) (BDH Laboratories Supplies, Poole, UK). The reducing buffer was added to the samples for 60 min at 60 °C followed by addition of alkylation buffer (10x of iodoacetomide in 55 mM ABC) to the samples. The samples were incubated in a dark place for 30 min at room temperature. Thereafter, samples were digested (1:50) by 20ng/μl of sequencing grade modified trypsin (Promega, Southampton). Later on, 10% acetonitrile (ACN) (BDH) was added to activate the trypsin digestion binding and the samples were incubated overnight at 37 °C. Digested samples were then dried in a vacuum centrifuge (miVac DNA concentrator, Barnstead Genevac, Ipswich, UK) for 30 min at 30^° C. The proteomic samples by this step were reduced, alkalyated and digested and ready for fractionation step.

The HK1 cells were seeded into 100-mm culture plates at a density of 4.0×10⁵ cells/dish in 6 ml culture medium. The cells were treated with GnRH at a concentration of 10^-9 M for 48 h. Untreated cells were used as a control. The cells were prepared for cell-cycle analysis using a CycleTEST PLUS DNA Reagent kit (BD BioSciences, San Jose, CA, USA). A pellet containing 5×10⁵ cells was gently resuspended in 250 μl solution A, containing trypsin (BD Biosciences), followed by 200 μl solution B, containing trypsin inhibitor and RNase A (BD Biosciences), and incubated at room temperature for 10 min each. A total of 200 μl propidium iodide (PI) was added and the cell suspensions were incubated at 4°C in the dark for 10 min. Flow-cytometric analysis of the cellular DNA content was performed using Cell Quest Pro software version 6.0 (BD Biosciences) on a FACS Calibur flow cytometer (BD BioSciences) and the results were analysed using ModFit LT™ software version 4.0 (Verity Software House, Inc., Topsham, ME, USA).

HeLa cells and astrocytes (a gift from Dr R Susuki, Photon Medical Research Center, Hamamatsu University School of Medicine) were cultured at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide (CO₂) in DMEM supplemented with 10% FBS and 100 units/ml penicillin-streptomycin. These cells were maintained in 60-mm dishes (Corning Incorporated, Corning, NY, USA). For studies of Ca²⁺ response, the cells were subcultured in 35-mm glass-bottom dishes (Iwaki Glass, Chiba, Japan) at approximately 0.5×10⁵ cells per dish.
Human umbilical vein endothelial cells (HUVECs), purchased from Health Science Research Resources Bank (HSRRB; Osaka, Japan), were maintained in E-STIM Endothelial Cell Culture Medium (BD Biosciences, Franklin Lakes, NJ, USA) with 10% FBS, 10 ng/ml EGF, and 200 µg/ml endothelial cell growth supplements (ECGS) on 75-mm flasks. They were cultured in a humidified incubator containing 5% CO₂ and 95% air at 37°C. The HUVECs were removed from the substrate with 0.25% trypsin and 0.02% EDTA (BD Biosciences) and passaged on coverslips in 35-mm-diameter culture dishes at a density of 1×10⁴ cells/ml. The subcultures were used for [Ca²⁺]_i measurements on day 2 after passage.