![Figure 5. NSAIDs inhibit TCR-dependent Fyn activation and Pyk2 phosphorylation. (A) Enolase phosphorylation in in vitro kinase assays of Fyn-specific immunoprecipitates from Jurkat cells or PBLs, nonactivated or activated by CD3 ligation for 1 minute, in the presence of carrier, 15 μM sc-560, or 250 μM NS-398. After the kinase reaction, samples were subjected to SDS-PAGE, transferred to nitrocellulose filters, and analyzed using a Phosphorimager. The filter was subsequently probed with anti-Fyn mAb as immunoprecipitation control. The data (representative of 2 independent experiments) show the fold variation in enolase phosphorylation in stimulated versus unstimulated samples. A representative autoradiograph showing [32P]-labeled enolase by Fyn in Jurkat cells is shown above the graph. The fold variation in Fyn autophosphorylation in CD3-stimulated versus unstimulated Jurkat cells was as follows: carrier, 1.95 ± 0.2-fold; sc-560, 1.05 ± 0.1-fold. (B) Lck autophosphorylation in in vitro kinase assays of Lck-specific immunoprecipitates from Jurkat cells, nonactivated or activated by CD3 ligation for 1 minute, in the presence of carrier or 15 μM sc-560. The samples were processed as in panel A. The data (representative of 2 independent experiments) show the fold variation in Lck autophosphorylation in stimulated versus unstimulated samples. A representative autoradiograph showing [32P]-labeled Lck is shown above the graph. (C) Immunoblot analysis of postnuclear supernatants from Jurkat cells, nonactivated (0) or activated by CD3 ligation (CD3) for 1 minute in the presence or absence of 15 μM sc-560 or 600 μM ibuprofen (ibupr). The filter was probed with antiphospho–Pyk2 antibodies, followed by anti-Pyk2 mAb as loading control. The migration of molecular mass markers is shown. (D) Enolase phosphorylation in in vitro kinase assays of Fyn-specific immunoprecipitates from Jurkat cells, nonactivated or activated by CD3 ligation for 30 seconds, in the presence of carrier or 1 μg/mL PGE2. The data from a representative experiment are shown, as well as the respective autoradiograph showing [32P]-labeled enolase. Error bars indicate standard deviation.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/105/5/10.1182_blood-2004-04-1299/6/zh80050574930005.jpeg?Expires=1735825488&Signature=bCLp2HwgOPNZq2ZxXLG11KBikaFc8XzbrvcvF461ss-wkW~fxhrhfmBAAiJXJURWtn16vJC7FFVfie5FL5LzeMMsJbEqGVVgOMRNiddrEBS4sjp~LV1vP1ChZsm6Wqx5q~04t5va6~25dBj7~UgHuriKq6VHEaESPdKwai~gqOwLChuS-vOgAv1NbnpeS3R~xIABeoFtzdtmo~7TErFfb91GQmsBr4ojlgDa-Do5mv7ELNlHjcplPGU5cu8VSDpVQuwzA2qwSLTyQR10LLHZ4n8ahAgDziXZPcHEqHC8pOV4UkRvB76XgvCk6dcEUXeYxh1a-yvibEbq4V3myfHbkA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
NSAIDs inhibit TCR-dependent Fyn activation and Pyk2 phosphorylation. (A) Enolase phosphorylation in in vitro kinase assays of Fyn-specific immunoprecipitates from Jurkat cells or PBLs, nonactivated or activated by CD3 ligation for 1 minute, in the presence of carrier, 15 μM sc-560, or 250 μM NS-398. After the kinase reaction, samples were subjected to SDS-PAGE, transferred to nitrocellulose filters, and analyzed using a Phosphorimager. The filter was subsequently probed with anti-Fyn mAb as immunoprecipitation control. The data (representative of 2 independent experiments) show the fold variation in enolase phosphorylation in stimulated versus unstimulated samples. A representative autoradiograph showing [32P]-labeled enolase by Fyn in Jurkat cells is shown above the graph. The fold variation in Fyn autophosphorylation in CD3-stimulated versus unstimulated Jurkat cells was as follows: carrier, 1.95 ± 0.2-fold; sc-560, 1.05 ± 0.1-fold. (B) Lck autophosphorylation in in vitro kinase assays of Lck-specific immunoprecipitates from Jurkat cells, nonactivated or activated by CD3 ligation for 1 minute, in the presence of carrier or 15 μM sc-560. The samples were processed as in panel A. The data (representative of 2 independent experiments) show the fold variation in Lck autophosphorylation in stimulated versus unstimulated samples. A representative autoradiograph showing [32P]-labeled Lck is shown above the graph. (C) Immunoblot analysis of postnuclear supernatants from Jurkat cells, nonactivated (0) or activated by CD3 ligation (CD3) for 1 minute in the presence or absence of 15 μM sc-560 or 600 μM ibuprofen (ibupr). The filter was probed with antiphospho–Pyk2 antibodies, followed by anti-Pyk2 mAb as loading control. The migration of molecular mass markers is shown. (D) Enolase phosphorylation in in vitro kinase assays of Fyn-specific immunoprecipitates from Jurkat cells, nonactivated or activated by CD3 ligation for 30 seconds, in the presence of carrier or 1 μg/mL PGE2. The data from a representative experiment are shown, as well as the respective autoradiograph showing [32P]-labeled enolase. Error bars indicate standard deviation.
