CXCL12 can activate ABL1 in T-ALL cells. (A,B) Starved JURKAT or CCRF-CEM cells were incubated with CXCL12, and then cell lysates were prepared: 500 μg of cell lysates were incubated with GST-Crk-CTD in the GST pull-down assay, followed by Western blot assay, using the antibody to ABL1. Western blot bands were analyzed using Image Pro Plus 6.0, and fold changes of active ABL1 were normalized according to GST-CRK-CTD. Total ABL1 was used as another loading control. (C) Next, 2 μg of GST-RhoGDI2-Y24F, GST-RhoGDI2 Y130F, or GST-RhoGDI2 Y153F were introduced and incubated with active ABL1 immunoprecipitated from CCRF-CEM cells treated with CXCL12 at 37°C for 30 min in the in vitro kinase buffer for 30 min. Samples were boiled in 1× Loading buffer at 98°C for 5 min and then resolved by SDS-PAGE. PY20, ABL1, or GST antibody was used in the Western blot assay to detect the phosphorylation of RhoGDI2, the amount of active ABL1, or RhoGDI2 (WT or mutated) that we used in each in vitro kinase assay, respectively. Phosphorylated RhoGDI2 were normalized according to the mutants of GST-RhoGDI2. ABL1 was used as another loading control.