174 Fig. 1 Guanosine-5′-O-(3-thio)triphosphate (GTP-+-S) is an effective chemorepellent in Tetrahymena. In vivo behavioral bioassays (see “Materials and methods”) were used to show the concentration dependencies for avoidance reactions to GTP-+-S. The percentage of cells showing avoidance was determined by observation of a single cell after transfer to the test solution. Each trial consisted of ten cells, which were individually scored as to whether or not avoidance oc- curred. Each point represents the mean ± standard deviation (SD) of at least three trials. Error bars, representing the standard deviation, are shown for each point. Minimum concentration required to give 100% avoidance was 100 μM for GTP-+-S. This concentration was used in the inhibition studies that follow. The EC50 of this compound was approximately 30 μM earlier, this finding is not unexpected. The EC50 of GTP-+-S in this study was approximately 30 μM. To determine whether a tyrosine kinase activity was involved in GTP avoidance, we used the tyrosine kinase inhibitor, genistein (Fig. 2). Genistein itself did not cause avoidance reactions, nor was it toxic to the cells. Baseline avoidance of ∼ 20% [9] to 100 μM GTP-+-S was seen at a genistein concentration of 100 μg/ml. The inhibition constant (IC)50 of genistein was approximately 40 μg/ml. Another phytoestrogen, daidzein, served as a control for nonspecific side effects of genistein. As seen in Fig. 2, this compound had no measurable effect on avoidance at the tested concentrations, suggesting that the inhibitory effect of genistein was indeed due to its tyrosine kinase inhibition and not attributable to some side effect of the drug. As an additional control for specificity, we tested whether genistein had any effect on the transduction of another chemorepellent, adenosine 5′-O-(3-thio)tri- phosphate (ATP-+-S). We used 150 μM ATP-+-S, a concen- tration that normally causes 100% avoidance in T. thermophila [12]. When cells exposed to ATP-+-S were first incubated for 10–15 min in 100 μg/ml genistein, no measurable effect on avoidance was seen. Cells continued to avoid 150 μM ATP-+-S at a rate of 96.6 + 5.8% (n ≥ 3). Genistein also failed to affect avoidance to the chemo- repellent pituitary adenylate cyclase-activating polypeptide (PACAP). When testing 0.1 μM PACAP, which normally Purinergic Signalling (2008) 4:171–181 causes 100% avoidance in T. thermophila [9], we found that prior incubation with 100 μg/ml genistein did not markedly affect avoidance. Cells continued to avoid 0.1 μM PACAP at a rate of 96.6 + 5.8% (n ≥ 3). These data suggest that tyrosine kinase activation is specifically related to GTP exposure rather than being a general response to chemorepellent exposure. In an attempt to localize the phosphotyrosines within the cell, immunofluorescence experiments were performed using a polyclonal antiphosphotyrosine antibody. Three separate trials were done with this antibody, and represen- tative photographs are compiled in Fig. 3a–c. GTP-exposed cells (Fig. 3b) showed higher overall fluorescence intensity than did the controls and genistein-treated GTP-exposed cells (Fig. 3a,c). Ciliary staining was seen in the GTP- exposed cells, along with punctuate staining of the cytosol. The control and genistein-treated GTP-exposed cells showed the same cytosolic staining pattern, but ciliary staining was not detected in these control groups, perhaps because the overall fluorescence level of these cells was so low. To determine which proteins were being phosphorylated by the tyrosine kinase in response to GTP-+-S, we also performed several Western blots of whole-cell extract obtained from control and GTP-+-S -treated cells using the polyclonal antiphosphotyrosine antibody as a probe. Protein extracts were standardized so that lanes were loaded equally, and a control, Coomassie-stained gel, was also run to control for lane-loading artifacts (data not shown). A representative Western blot is pictured in Fig. 3d. Both GTP-treated and control cells showed similar staining at Fig. 2 The tyrosine kinase inhibitor, genistein, blocks avoidance to 100 μM guanosine-5′-O-(3-thio)triphosphate (GTP-+-S) in Tetrahy- mena. The phytoestrogens genistein (closed circles) and daidzein (open circles) were used as inhibitors of avoidance. Baseline avoid- ance was seen at a genistein concentration of 100 μg/ml. The IC50 of genistein was approximately 40 μg/ml. Daidzein served as a con- trol for nonspecific effects of genistein and had no measurable effect on avoidance at the concentrations tested. Data shown represent the mean +/− standard deviation (SD) for three or more trials