PI3K and Akt are activated in DCs stimulated with CCL19 and CC21 and regulate apoptosis. (A) DCs suspended in 0.1% BSA in RPMI that were untreated (control) or pretreated with LY294002 (100 μM) for 60 minutes (LY294002) were left unstimulated (–) or were stimulated (+) with CCL19 or CCL21 for the indicated times. Cells were lysed, PI3K was precipitated, and in vitro kinase (IVK) performed as described in “Materials and methods.” PIP indicates PtdIns-3-phosphate. In parallel experiments, immunoprecipitates were also analyzed by Western blotting (WB) to show equal levels of PI3K. A representative experiment out of 4 performed is shown. (B) Whole-cell lysates of cells stimulated with CCL19 or CCL21 for the indicated times were separated on SDS-PAGE and transferred to PVDF membranes for subsequent Western blotting. Activated Akt was detected with an antibody reacting with phosphorylated P-Ser 473 (p-Akt). To confirm equal loading, blots were reprobed with an antibody reacting with total Akt1. (C) DCs suspended in 0.1% BSA in RPMI were left untreated (control) or pretreated with LY294002 (100 μM) for 60 minutes. Then, DCs were either left unstimulated (–) or stimulated (+) with CCL19 or CCL21. (Bottom) Western blots. After 2.5 minutes of stimulation with chemokines, aliquots of DCs were taken to analyze the level of phosphorylated/active Akt1 (p-Akt) and total Akt1 by Western blotting. A representative experiment out of 3 performed is shown. (Top) Bar diagrams. Remaining DCs were left for an additional 6 hours, and then the percentage of annexin V–positive/7-AAD–negative cells was quantified. The results represent the mean ± SEM of 3 independent experiments.