Src activity involvement in DEX-induced thymocyte apoptosis.
Effect of PP1 on DEX-induced PI-PLC activity, apoptosis, GR assembly, and GR nuclear translocation. (A) Thymocytes were pretreated for 30 minutes with PP1 (10 μM), U73122 (2.5 μM), and D609 (50 μg/mL) and then with DEX (10−7 M, 5 minutes). To evaluate PI-PLC activity, cell extracts were reacted with radiolabeled PI vesicles, and then the DAG released was separated by TLC and visualized by autoradiography. *P < .001. (B) Effect of PP1, U73122, and D609 on DEX-induced thymocyte apoptosis, as detected after 18 hours of culture with DEX (10−7 M). Apoptosis was evaluated by PI staining and FACScan flow cytometer. Mean values ± SE of 3 different experiments in duplicate are reported. *P < .001. (C) Thymocytes were pretreated for 30 minutes with PP1 (10 μM) before treatment with DEX (10−7M, 30 minutes). Cell lysates were immunoprecipitated (IP) with anti-HSP90 antibody and then Western blotted with anti-HSP90, anti-GR, and anti-Src antibodies. (D) Immunofluorescence analysis of thymocytes untreated or treated for 30 minutes with DEX alone or in combination with PP1 and U73122. After treatment, cells were stained with anti-GR antibody using the paraformaldehyde-saponin procedure.
