DEX inhibits signaling pathways downstream of the TCR. Cells were preincubated for 10 minutes with or without DEX and subsequently stimulated for 15 minutes using anti-CD3 and anti-CD28 Abs. The phosphorylation status of Fyn, Src, LAT, PKB, PKC, p38, ERK1/2, and JNK in whole-cell lysates was assayed using phosphospecific mAbs on Western blot. An Ab against phosphorylated Lck associated with increased activity (not available) was not included.48 The Ab we used to detect phosphorylated Src may cross-react with other Src kinase family members, including Fyn, Lck, Lyn, pp62c-yes (Yes), hematopoietic cellular kinase (Hck), and Src itself when phosphorylated at equivalent sites. Therefore, we cannot exclude the possibility that suppressed Src phosphorylation represents reduced phosphorylation of Src itself or other Src family members. To test for equal loading Western blots were stripped and reprobed using the appropriate Abs. The Western blots represent 3 independent experiments. LAT indicates linker for activation of T cells; PKC, protein kinase C; PKB, protein kinase B; DEX, dexamethasone.
