Figure 1.

Adenosine activates TrkA receptors at multiple phosphotyrosine residues. A, PC12 cells were treated with 10 μm adenosine for the indicated times. Trk was immunoprecipitated from cell lysates using a pan-Trk antibody. Phosphorylation of Trk was assessed with antibodies against phosphotyrosine 499 of rat TrkA [pTrk 490 (Cell Signaling)] or antibodies against phosphotyrosines 683 and 684 of rat TrkA [pTrk 674/675 (Cell Signaling)]. Immunoblot with pan-Trk antibody (B3) was used to ensure equal loading. B, PC12 cells were pretreated with either 20 μg/ml cycloheximide or 200 ng/μl actinomycin D for 30 min before incubation with 10 nm CGS 21680 for 1 or 3 hr. Trk was immunoprecipitated from cell lysates and analyzed with phosphotyrosine antibodies. Trk protein levels were determined by blotting with pan-Trk antisera. C, Quantitation of phospho-Trk and total Trk levels from lysates of CGS 21680-treated PC12-615 cells. Three independent experiments were quantified using ImageJ analysis software. Levels of band intensity relative to untreated control are represented on the y-axis. Error bars reflect SEM. D, RT-PCR for NGF mRNA was performed on total RNA extracted from untreated PC12 cells or cells treated with 10 nm CGS 21680 for 3 hr. RNA extracted from adult rat submandibular gland was used as a positive control; GAPDH was used as an internal loading control.