Fig. 1.

AFKs are present in ciliary ganglion homogenates and neurons, and Abl kinase activity is inhibited by STI571. A, developmental expression of AFKs. Homogenates prepared from ciliary ganglia (0.15 mg/ml total protein) throughout the developmental period when nicotinic synapses form and mature (E6, -8, -11, and -14) were subjected to immunoprecipitation with (+) or without (−) anti-Abl K-12. After probing with anti-Abl mAb8E9, an interacting protein expected at ∼145 kDa (arrow) was detected at each developmental age. B, quantification reveals that Abl family kinase levels progressively increase between E6 and E14. Results are expressed as band intensity (AU) per milligram of ganglionic protein loaded (□) or as band intensity per neuron at the indicated embryonic age (■) (mean ± S.D.). C, neuronal localization. Confocal images (single 1-μm optical sections) depict an acutely dissociated E14 CG neuron (left) and a CG neuron grown 4 days in culture (right) immunolabeled using anti-Abl K12 antibody and reveal a largely cytoplasmic distribution of AFKs. Control neurons (no added primary antibody) were unlabeled (data not shown). Scale bar, 15 μm. D, STI571 inhibits endogenous Abl kinase activity. E14 ciliary ganglion quadrants were pretreated with (+) or without (−) STI571 (10 μM; 30 min); lysates were then prepared, immunoprecipitated with anti-Crk, and probed either with anti-Crk (left) or anti-pY221Crk (right). Note that pTyr221 levels are undetectable in lysates from ganglia after pretreatment with STI571. Each lane represents 40 CG equivalents, or approximately 14.3 mg total protein (wet weight). The arrow indicates 37 kDa. Similar results were obtained from two other experiments.