Figure 2.

Analysis of mutants. Cell lines were stained with PAC1 in the presence (open histograms) or absence (filled histograms) of 2 μM Ro43-5054. (a) All mutant cell lines failed to bind PAC1 (filled). Note that in the example shown, PAC1 fluorescence is superimposable on PAC1 fluorescence in the presence of the competitive inhibitor, Ro43-5054 (open). (b) Mutant cell lines were retransfected with wild-type α_IIbα_6Aβ₃β₁ and Tac-α₅. Cells were double-stained with PAC1 and anti-Tac 48 hr later. Depicted is PAC1 staining on the gated subset of Tac-positive cells. In some transfected mutant cell lines, PAC1 binding was restored, indicating that the defect was in the integrin. (c and d) Mutant cell lines were stained with PAC1 as described in a. In these assays, anti-LIBS6 was added at the same time as PAC1. With the addition of anti-LIBS6, some mutant cell lines still failed to bind PAC1 (c), indicating a ligand binding-defective phenotype. In others, anti-LIBS6 restored PAC1 binding (d), indicating an activation defect.