SLAP and SLAP2 inhibit GPVI/FcRγ and CLEC-2 signaling in the DT40 B-cell line model. (A) The DT40 B-cell line was transfected with a Ca2+/ mitogen-activated protein kinase-responsive NFAT/AP-1-luciferase reporter construct, a β-galactosidase construct, a GPVI, an FcRγ, a SLAP or SLAP2 expression construct, or empty vector control. Cells were either left unstimulated or stimulated with 5 μg/mL collagen, lysed, and assayed for luciferase and β-galactosidase. Luciferase data were normalized for β-galactosidase values. (B) The expression of GPVI was confirmed by flow cytometry with an anti-GPVI mAb. (C) The experiment was conducted as described in (A), with the exception that cells were transfected with a myc-tagged CLEC-2 expression construct and stimulated with 50 nM rhodocytin. (D) The experiment was performed as for (B), except that CLEC-2 was detected by an anti-myc mAb. Results in all panels are expressed as mean ± standard deviation (SD) (n = 3). ***P < .001. ctrl, control; RC, rhodocytin; unstim., unstimulated.
