P akt s473 alexa fluor 647 d9e

After incubation with 50 ng/ml CCL22 for the indicated time, cells were fixed in 2% paraformaldehyde for 10 min at room temperature and then permeabilized with cold methanol at −20°C overnight. Cells were washed twice in FACS buffer and stained with p-Akt (S473)–Alexa Fluor 647 (D9E) or isotype control (DA1E; Cell Signaling Technology) for 20 min at room temperature. Cells were washed and resuspended in FACS buffer for analysis on a FACSCalibur.

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.