DC-SIGN/Fc binding to FL receptor V-region mannosylation is not sufficient to induce Ca²⁺ influx. (A-B) Lectin binding to transduced TKO cells in lectin buffer either in the absence (A) or in the presence (B) of Ca²⁺ was measured by FACS. (A) Gray shaded area represents binding to empty-vector–transduced cells in absence of Ca²⁺; dotted line represents binding to TKO cells expressing the indicated receptors in absence of Ca²⁺. (B) Gray shaded area shows binding to empty-vector–transduced cells in the presence of Ca²⁺; black line represents binding to receptor-positive cells in the presence of Ca²⁺. (C) FACS analyses of Ca²⁺ flux of TKO cells transduced with ERT2-SLP65 and FL receptors in lectin buffer containing 10 mM Ca²⁺. After 1-minute baseline measurement, cells were stimulated with OHT and DC-SIGN/Fc that was preincubated with anti-IgG for further multimerization. Anti-μHC stimulation served as control. (D-E) Lectin binding and Ca²⁺-flux measurements were carried out in medium supplemented with 1% FCS and physiological Ca²⁺-ion concentration. (D) Gray shaded area represents binding to empty-vector–transduced cells in medium; black line represents binding to TKO cells expressing the indicated receptors in medium (D) FACS analyses of Ca²⁺ flux of TKO cells transduced with ERT2-SLP65 and FL receptors in medium containing 1% FCS as described in (C). Results are representative of >3 independent experiments. EV, empty vector.