LIME is required for BCR-mediated activation of p42/44 MAPK, calcium flux, and NF-AT. (A) A20 cells were transfected with the control vector or plasmids expressing LIME siRNA along with pEGFPN-1. Forty-eight hours after electroporation, transfection efficiency was analyzed by FACS (left panels). The reduction of LIME expression upon expression of siRNA was assessed by Western blotting with an anti-LIME Ab (right panel). (B) LIME mediates BCR-dependent MAPK activation. A20 cells were transfected with pSUPER or pSUPER encoding siRNA directed against murine LIME. After 48 hours, transfected cells were activated with 20 μg/mL F(ab′)₂ goat anti-mouse IgG for 1 and 3 minutes at 37°C. Lysates were analyzed by Western blotting with an anti-phospho-ERK or anti-ERK Ab. (C) LIME is required for the BCR-mediated Ca²⁺ response. A20 cells were transfected with pSUPER vector or pSUPER expressing an siRNA against LIME. After 48 hours, transfected cells were loaded with fura-2/AM for 30 minutes at 37°C. Subsequently, cells were stimulated with 20 μg/mL F(ab′)₂ goat anti-mouse IgG, and the intracellular calcium level was measured. (D) LIME mediates BCR-dependent NF-AT activation. The plasmids expressing siRNA against LIME were cotransfected into A20 cells with an NF-AT-luciferase reporter construct. After 48 hours, the cells were incubated for 6 hours with medium alone, F(ab′)₂ goat anti-mouse IgG, or PMA/ionomycin, and then harvested. Subsequently, the cells were lysed and the luciferase activity was assayed. To control for the transfection efficiency, the luciferase activities of medium or F(ab′)₂ goat anti-mouse IgG-treated samples were normalized to those of PMA/ionomycin-treated samples. The experiments were performed twice in triplicate. Error bars indicate SD.