Stable expression of GPVI by Dami cell lines. (A) The requirement for exogenous FcRγ was tested by comparing the expression of transfected wild-type GPVI in cell lines that were not cotransfected with FcRγ (1G4) or cotransfected with FcRγ (1C2 and 1E3). The binding of biotin-CVX to GPVI in lysates from each cell line indicate that 1G4 expresses as much or more total GPVI as do 1C2 or 1E3. (B) Adhesion of Dami cell lines to CVX (▪), CRP (□), control peptide GPP (▧) or control peptide GPA (▨). Optical density (OD) is indicated on the abscissa. The identity of each cell line is indicated on the ordinate. Values represent mean ± SD for 3 experiments. (C) Immunoprecipitation. Rabbit polyclonal anti–human FcRγ was used to precipitate endogenous FcRγ from 1G4. The precipitated proteins were isolated, solubilized, and analyzed by Western blot using either biotin-CVX (lane 1) or the same polyclonal anti–human FcR antibody (lane 2) to visualize the proteins. The electrophoretic mobilities of selected molecular weight marker proteins are indicated to the left of the gel. (D) Adhesion of Dami lines transfected with wild-type GPVI (WT) or GPVI substitution mutants N92A, S94A, and L95H (ordinate). Adhesion to plates coated with BSA (▦), CVX (▪), or CRP (□) was measured. Optical density (OD) is indicated on the abscissa. Error bars represent 1 SD.