Purinergic Signalling (2008) 4:171–181 small, tight circles; sudden reversal of swimming direction; or any deviation from the normal helical swimming pattern of T. thermophila. Western blots Western blots were performed as follows: For each sample, 10 ml of the same 2-day-old culture was washed 3× in behavioral buffer; 100 μM GTP-+-S (the concentration required to elicit 100% avoidance) was added to the tube of cells marked GTP. The control tube received only buffer; no GTP was added. Both tubes were immediately extracted with 0.1% sodium dodecyl sulphate (SDS) in the presence of 1 mM sodium orthovanadate for 30 min at 4°C. Extracts were spun at 16,000 g at 4°C for 30 min to precipitate insoluble components. Supernatant was collected, and 5× loading buffer was applied in preparation for SDS poly- acrylamide gel electrophoresis (PAGE); 100 μl of each sample was applied to a 10% SDS-PAGE. Amount of protein loaded onto the gel was standardized using the BIO- RAD protein assay; in addition, a control, Coomassie- stained gel, was run to show that equal amounts of protein were loaded into each well. Proteins were transferred from the SDS-PAGE to nitrocellulose for 30 min at 150 mA. Blots were then blocked overnight in a 3% solution of powdered milk. Blots were incubated with a 1:1,000 dilution of polyclonal phosphotyrosine antibody in the presence of 3% powdered milk for 1.5 h, washed 3× in TBS, then incubated with a 1:100 dilution of alkaline phosphate conjugated anti-rabbit antibody for 1.5 h in the presence of 3% powdered milk. Blots were then washed 3× with TBS and incubated for 5 min in an alkaline phosphatase buffer containing 100 mM NaCl, 5 mM MgCl2, 100 mM Trizma base, pH 9.5 with hydrogen chloride (HCl). Alkaline phosphatase buffer was then removed, and blots were developed using the Sigma-Fast system until bands were visible. EIA assays NO assays were carried out using 2-day-old cell cultures. Cells were washed twice in buffer and diluted to a final concentration of approximately 720 cells/ml. Cells were exposed to 100 μM GTP-+-S for approximately 30 s, then spun down in a bench-top microcentrifuge for 30 s. Control cells were spun under identical conditions except that no GTP was added to the solution. Supernatant from both sets of cells was analyzed for NO using a kit from BIOMOL according to the manufacturer’s instructions. cGMP assays were carried out using 2-day-old cell cultures. Cells were concentrated to approximately 7.66× 106 cells/ml in order to maximize cGMP production. Cells were exposed to 100 μM GTP for approximately 30 s, then 173 immediately lysed by freezing in liquid nitrogen and thawing. Theophylline (1 mM) was added to the lysate to inhibit phosphodiesterases. Lysate was spun at 16,000 g for 30 min. Control cells were treated in identical fashion except for the addition of GTP. Supernatant from both sets of cells was assayed for cGMP using a kit from Cayman Chemicals according to the manufacturer’s instructions. Immunofluorescence Ten milliliters of 2-day-old cells were starved overnight in behavioral buffer then washed 3× in behavioral buffer and concentrated down into 2 ml. Mucocysts were removed by adding Alcian Blue GX to a final concentration of 0.05%, then adding nine volumes of 0.25% proteose peptone [11]. Mucocysts were allowed to precipitate by standing the tube in a rack for 10 min. Supernatant (including cells) was removed from the top of the tube. The cells were then washed 3× in behavioral buffer to remove any remaining mucocysts or Alcian Blue and concentrated down into 3 ml total; 1 ml of cells was set aside as the control cells. Another milliliter of cells was incubated with 100 μM GTP-+-S. A third aliquot was incubated with 100 μg/ml genistein prior to GTP treatment. Immediately, all three aliquots of cells were fixed in 3.7% formaldehyde (from 37% stock diluted into behavioral buffer) with gentle shaking for 20 min. Cells were washed 3× in 1× TBS/ NP40 (Modern Biology) then incubated with a 1:100 dilution of polyclonal antibody in 1× TBS/NP40 for 1.5 h. Cells were again washed 3× in 1× TBS/NP40 then incubated in a 1:100 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (ICN Biomedicals) for 1.5 h. Cells were washed 3× in 1× TBS before being viewed under a Nikon microscope at 400×. Cells were photographed using a SPOT digital camera using a 10-s exposure for each photograph. Results GTP-+-S is a chemorepellent in Tetrahymena,, with a minimum concentration of 100 μM required for 100% avoidance (Fig. 1). This concentration was used in the inhibition studies that follow as well as in our biochemical studies. We used GTP-+-S in our experiments rather than GTP to minimize the effects of the ecto-ATPase upon the assays [3]. However, the activity of apyrases and other ectoenzymes may still have degraded some of the GTP-+-S before it could reach its intended receptor. The concentra- tion of GTP-+-S required in order to achieve 100% avoidance in this study was 5× higher than that previously published [1]; however, considering that the cell line and growth conditions were different than those published