FIGURE 5.

CXCL12 can activate Src in T-ALL cells. (A,B) Starved JURKAT or CCRF-CEM cells were incubated with CXCL12, and then cell lysates were prepared: 60 μg of cell lysates were subject to SDS-PAGE and Western blot assay, using active Src antibody, Src(416Tyr-P). (C) Then, 1.2 μg of GST-RhoGDI2-WT, GST-RhoGDI2-Y24F, GST-RhoGDI2 Y130F, or GST-RhoGDI2 Y153F were incubated with 600 ng recombinant human active Src (His-tag) in the in vitro kinase assay. Samples were boiled in 1× Loading buffer at 98°C for 5 min and then resolved by SDS-PAGE. PY20, His antibody, or GST antibody was used in the Western blot assay to detect the phosphorylation of RhoGDI2, the amount of active Src, or RhoGDI2 (WT or mutated) that were used in each in vitro kinase assay, respectively. Western blot bands were analyzed using Image Pro Plus 6.0. Fold changes of active Src were normalized according to total Src. Bands of phosphorylated GST-RhoGDI2 (PY20) were normalized according to WT or mutants of GST-RhoGDI2 (GST) that were used in each kinase assay.