DEX-induced inhibition of Lck and Fyn kinase activities is GR dependent. (A) GR-negative Jurkat cells (confirmed by Western blot analysis; not shown) were pretreated with DEX for 10 minutes and incubated with anti-CD3, anti-CD28 mAbs for 15 minutes. Whole-cell lysates were used for Western blot analysis. Because there are no Abs commercially available targeting phospho-Lck associated with increased activity, phospho-ZAP70 expression (a downstream target of Lck) was analyzed. DEX treatment did not affect phospho-Fyn expression, nor did it affect phospho-ZAP70 protein levels in GR-negative Jurkat cells. Three experiments were performed, and similar outcomes were obtained. (B) CD4⁺ T cells were incubated for 1 hour with a GR mimetic (RU486; 50 μM), which acts as a GR agonist at this concentration. Subsequently, cells were treated with DEX (10 minutes) and activated with anti-CD3, anti-CD28 Abs (15 minutes). Lck and Fyn immunoprecipitates were subjected to in vitro kinase assay, and phospho-SAM68 expression was studied on Western blot using PY20. Decreased phospo-SAM68 expression was seen in Lck and Fyn immunoprecipitates prepared from activated cells treated with DEX, and cells treated with RU486 together with DEX. Incubation with RU486 alone (at this high/agonistic concentration) also resulted in impaired SAM68 phosphorylation. DEX indicates dexamethasone; IB, immunoblotting; IP, immunoprecipitation.