DEX inhibits Lck and Fyn kinase activity in vitro. (A) CD4⁺ cells were pretreated with a specific Src family kinase inhibitor (SU6656) for 45 minutes and subsequently incubated with DEX or DMSO supplemented media (control) for 10 minutes and activated with anti-CD3 and anti-CD28 Abs for 15 minutes. In vitro kinase assays were performed using SAM68 as a substrate, and Src-like kinase activity was analyzed on Western blot using PY20. Equal loading was evaluated with an Ab against actin. Phospho-SAM68 expression is observed upon TCR ligation. Incubation with SU6656 resulted in abrogation of SAM68 phosphorylation, and a similar effect was demonstrated in activated cells treated with DEX. (B) Lysates of CD4⁺ cells, pretreated with DEX (10 minutes) and subsequently activated (15 minutes), were subjected to Lck and Fyn immunoprecipitation, followed by in vitro kinase assay. To test for equal loading, Western blots were analyzed for total Lck and Fyn expression. Reduced phospho-SAM68 expression was seen in activated cells incubated with DEX, compared with cells incubated in the absence of DEX. Similar results were obtained in 3 independent experiments. DEX indicates dexamethasone; IB, immunoblotting; IP, immunoprecipitation.