PTEN-null Jurkat T cells exhibited potent chemotactic response to SDF-1α. (A) Jurkat cells were PTEN deficient. Lanes 1 to 6 contained lysates of wild-type Jurkat cells obtained from the laboratories of Drs Ji-Ming Wang, William L. Farrar, and Daniel W. McVicar, as well as our lab (all at NCI-Frederick, Frederick, MD), respectively, and the Tet-on and Tet-off Jurkat T-cell lines purchased from Clontech (Palo Alto, CA). Lanes 7 to 9 showed the lysates of HEK293 cells and PBMCs, used as positive controls. Cultured cells were lysed and electrophoresed on an SDS–polyacrylamide gel electrophoresis (PAGE) gel, transferred to nitrocellulose membranes, and probed with anti-PTEN or anti-ERK1/2 as a loading control. (B) Jurkat cells express CXCR4. The surface expression of chemokine receptor CXCR4 in Jurkat cells was measured by flow cytometry. Cells were stained with anti-hCXCR4 (solid line) or isotype-matched human IgG2a as a negative control (dotted line). (C) Jurkat cells exhibit dose-dependent chemotactic responses to SDF-1α. Chemotaxis was performed using 48-well chemotaxis chambers as described in “Materials and methods.” Different concentrations (0-1000 ng/mL) of SDF-1α were used for chemotaxis. (D) SDF-1α–induced chemotaxis of Jurkat cells was PTX sensitive. Jurkat cells were pretreated with different doses of PTX for 30 minutes at room temperature (RT) and chemotaxis assay was carried out as described in “Materials and methods”. All the results in this figure were representative of at least 3 independent experiments. (C-D) Data shown are means ± SD.