DM induces Lck phosphorylation in resting T cells but not activated T cells in a GCR-dependent fashion. (A) Resting human T cells (top panel) and Jurkat cells [E6.1] (bottom panel) were treated with CXCL12 (100 ng/mL), DM (1 μM), or the combination for 5 minutes. In the activated T-cell cultures, primary human T cells were incubated with 5 μg/mL antihuman CD3 mAb in combination with 5 μg/mL antihuman CD28 antibody for 1 minute at a cell concentration of 5 × 10⁶ cells/mL followed by treatment with CXCL12 and DM alone or with combination for 5 minutes. Treated cells were subsequently washed with ice-cold phosphate-buffered saline and then lysed with ice-cold radioimmunoassay (RIPA) buffer. Total Lck was immunoprecipitated from the lysates and phospho- and total Lck levels were assessed by immunoblotting using the phosphotyrosine and total Lck antibodies. In many of the gels, densitometry was performed on scanned gel images and assessed by UN-SCAN-IT gel digitizing software (Silk Scientific, Orem, UT). The data were then normalized and presented as fold change over control bands. Here, phospho-Lck was normalized against the corresponding band of total Lck. The final data are presented as the fold change over the control (P/T). (B) Primary human T cells were incubated in the presence or absence of 5 μM mifepristone (RU486) for 1 hour. The cells were then treated with CXCL12 (100 ng/mL) or DM (1 μM) or together for 5 minutes. Lysis of the cell and rest of the procedure for Lck immunoblot was followed as described in Figure 1A.