Functional assays in CXCR4 variants: enhanced receptor activation and prolonged intracellular signaling. (A) cAMP production was determined in stable NALM6 cells expressing WT CXCR4 (CXCR4^WT) or CXCR4 variants, after 30 minutes of stimulation in the presence of CXCL12; data represent mean ± standard error of the mean (SEM) of 4 independent experiments. (B) NALM6 cells were serum starved for 4 hours and stimulated with 100 nM rhCXCL12 for 10, 20, or 30 minutes, and ERK1/2 phosphorylation (T202/Y204) was measured by flow cytometry and represented as mean fluorescent intensity (MFI) fold change from baseline. Values represent mean ± SEM of 5 independent experiments. Comparisons are made to WT. ∗P < .05 (2-tailed unpaired Students t test). (C) Calcium mobilization was determined in WT and CXCR4 mutant NALM6 cells. Measurements were taken every second before and after ligand binding, for 2 minutes. Values represent mean ± SEM of 3 experimental triplicates of 5 independent experiments. (D) Right panel shows the area under the curve (AUC) of calcium mobilization, calculated for each cell line. ∗P < .05; ∗∗P < .01 (2-tailed unpaired Student t test). (E) Chemotaxis assay in transwells. Data show the fold of migrated cells to the lower chamber for 4 hours compared with WT. (F) NALM6 cells were serum starved for 4 hours and stimulated with 100 nM rhCXCL12 for 10, 20, or 30 minutes, and AKT phosphorylation (S473) was measured by flow cytometry and represented as MFI fold change from baseline. Values represent mean ± SEM of 5 independent experiments. Comparisons are made with WT. ∗P < .05; ∗∗P < .01 (2-tailed unpaired Student t test).