Purinergic Signalling (2008) 4:171–181 175 Fig. 3 Guanosine 5'-triphosphate (GTP) exposure increases tyrosine kinase activity in Tetrahymena. a–c Immunofluorescence labeling of phosphotyrosines in Tetrahymena is evidence of tyrosine kinase activity. Control (a), GTP-exposed (b), and genistein-treated GTP- exposed cells (c) were fixed and labeled with a polyclonal antiphosphotyrosine antibody. GTP-exposed cells showed higher fluorescence intensity than did control cells and cells treated with the tyrosine kinase inhibitor genistein. In addition, GTP-exposed cells showed ciliary staining in addition to punctuate staining of the cytosol. Total magnification 400×. Cell length ∼ 50 μm. d Western blot of whole-cell extract obtained from control and GTP-exposed cells using a polyclonal antiphosphotyrosine antibody shows increased phosphor- ylation levels in extract taken from GTP-exposed cells (lane 2) relative to extract from control cells (lane 3). A 66-kDa phosphopro- tein stained similarly in both the GTP and control lanes, whereas bands of 42, 35, and 21 kDa were more heavily stained in the GTP lane relative to the control lane. Molecular weight markers are shown in the first lane (molecular weights in kDa). A Coomassie-stained sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE) run as a loading control showed equivalent staining of proteins in all lanes (not shown) 66 kDa. However, increased phosphotyrosine levels were seen in the GTP-+-S-treated cells at 42, 35, and 21 kDa, consistent with tyrosine kinase activity. These bands were also present in the control cell extract; however, the phosphorylation level in these lanes was so low that they were barely detectable with our staining procedure. Addi- tional bands were also seen in both the GTP-exposed and the control lanes; however, they were also too light to be measurable. A more sensitive assay, such as chemilumines- cence based Western blot detection, might help to give a more complete profile of the phosphotyrosine-containing proteins shown in this procedure.