Opposite effect of IL-15 and IL-2 on CX3CR1 expression in cytokine-dependent mouse bone marrow-derived cells. (A) Total cellular CX3CR1 protein. Wild-type C57Bl/6 mouse bone marrow cell differentiation was initiated ex vivo for 3 days in IL-15 for all cultures. Cells were then washed and incubated in the presence of IL-2 (100 U/mL) or IL-15 (100 ng/mL) for an additional 11 days, giving a total of 14 days of cytokine treatment. Cells were also treated for a total of 14 days with a combination of the 2 cytokines according to the following protocol: IL-15 for 3 days → wash → IL-15 for 7 days → wash → IL-15 plus IL-2 for 4 days. Protein extracts were analyzed by Western blot using antibodies for CX3CR1 or β-actin. Control lanes contained a commercial source of protein extracts from C57Bl/6 mouse skeletal muscle and brain. (B) RNA analysis. Total RNA was isolated from bone marrow cells grown according to exactly the same protocol as in panel A. To control for specificity, cells were also cultured according to this protocol from both CX3CR1-/- knock-out (ko) and wild-type (wt) mice. RT-PCR using primers for the genes listed at the lower left of each panel was performed. (C) Densitometry of results shown in panels A and B. The results shown are from a single experiment representative of at least 3 separate experiments.