Human gamma globulin fraction 2

Doxycycline, puromycin, ethidium bromide, and Lipofectamine 2000 were purchased from Invitrogen. Recombinant human CCL17/TARC and recombinant human CCL22/MDC were purchased from R&D Systems. Human recombinant IL2 was obtained from Roche. Anti–human CCR4 antibodies (clone 1G1) conjugated to PE and Alexa Fluor 647 fluors were purchased from BD. The following antibodies were purchased from Cell Signaling Technology: Akt, P-Akt (Ser473; D9E), P-Akt (S473)–Alexa Fluor 647 (D9E), and Alexa Fluor 647–conjugated isotype control antibody (DA1E). Anti–rabbit IgG-HRP antibody was purchased from GE Healthcare. CD8a(Lyt 2) microbeads were purchased from Miltenyi Biotec. BKM120 was purchased from Selleck Chemicals. PTX was purchased from Sigma-Aldrich. Big Dye Terminator v1.1 was purchased from Applied Biosystems. Human gamma globulin fraction II was purchased from ICN Biomedicals Inc. Paraformaldehyde was purchased from Electron Microscopy Sciences.

Cells were washed with PBS containing 200 µg/ml of human gamma globulin (Human Gamma Globulin Fraction II; ICN Biomedicals Inc.) twice and incubated for 30 min on ice with anti–human CCR4-PE or CCR4–Alexa Fluor 647 (1G1; BD) or control isotype IgG. After washing with FACS buffer (PBS/1% FCS) twice, the cells were analyzed on a FACSCalibur (BD). For the CCR4 internalization assay, cells were exposed to CCL22 for varying lengths of time and were then treated for 1 min at 37°C to an acid buffer (pH 3.0, consisting of 50 mM glycine and 100 mM NaCl) to remove cell-bound CCL22. The cells were washed twice and then stained with anti–human CCR4–Alexa Fluor 647 on ice for 30 min, fixed with 2% paraformaldehyde (Electron Microscopy Sciences) on ice for 10 min, and analyzed on a FACSCalibur.