Gi and βγ subunits regulate apoptosis and activation of Akt. (A) DCs were left untreated (control) or pretreated with PTX (100 ng/mL) for 120 minutes. Subsequently, DCs were left unstimulated (–) or stimulated (+) with CCL19 or CCL21. (Bottom) After 2.5 minutes of stimulation with chemokines, aliquots 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) Remaining DCs were left for an additional 6 hours, and then the percentage of annexin V–positive/7-AAD–negative cells was quantified. Fluorescence-activated cell sorter (FACS) analysis performed in parallel showed that PTX treatment did not affect the levels of CCR7 (not shown). The results represent the mean and SEM of 3 independent experiments. (B) DCs were transfected either with vector or with βARK-CT. (Top) Eighteen hours after transfection, aliquots of vector- and βARK-CT–transfected DCs were taken to analyze βARK-CT levels by Western blotting using an anti-βARK antibody.28 β-actin levels show equal loading of the gels. (Bottom) Vector- and βARK-CT–expressing DCs were washed and subsequently incubated for 6 hours in 0.1% BSA in RPMI without (–) or with (+) CCL19 or CCL21. The percentage of annexin V–positive/7-AAD–negative cells was determined. A representative experiment out of 3 performed is shown.
