Figure 3.
Ablation of the αIIb C490-C545 disulfide bond restricts the distribution of αIIbβ3 to focal adhesions. (A) BHK cells were transfected with WT β3 and either WT or cysteine mutant αIIb and cells sorted for high β3 expression. Expression of β3 in the clones used for the experiments is shown as a bar graph (i) or representative histogram (ii). The bars and errors are mean ± standard error of the mean of 6 biological replicates. (B) Ablation of the αIIb C490-C545 disulfide bond reduces adhesion of cells to immobilized fibrinogen but not poly-D-lysine. The bars and errors are mean ± standard deviation (SD) of 4 independent experiments with 1 or 2 biological replicates. (C) WT or cysteine mutant αIIb-expressing BHK cells were seeded onto a fibrinogen-coated surface in the presence of β3 mAb VI-PL2 and allowed to adhere for 1 hour. After fixation, cells were stained for vinculin (7F9) and nuclei (DAPI), before being imaged using PerkinElmer’s Opera Phenix High-Content Screening System at ×63 magnification in confocal mode. Scale bar, 50 µm. (D) Integrin clusters were quantified in cells seeded onto poly-D-lysine or fibrinogen and expressed per cell (i) or per square micrometers (ii). Discreet integrin clusters in cells adhered to fibrinogen were more abundant when the αIIb C490-C545 disulfide bond is missing. The bars and errors are mean ± SD of 4 to 8 biological replicates. (E) WT or cysteine mutant αIIb-expressing BHK cells were seeded onto a fibrinogen-coated surface in the presence of β3 mAb VI-PL2 and allowed to adhere for 1 hour. After fixation, cells were stained for vinculin (7F9), phosphorylated FAK (pFAK, phosphoY397 FAK mAb), and nuclei (DAPI ), before being imaged by using PerkinElmer’s Opera Phenix high-content screening system at ×63 magnification in confocal mode. Scale bar, 50 µm. (F) F-actin and pFAK intensity in cells seeded on the fibrinogen-coated surface are expressed per µm2. The bars and errors are mean ± SD of 3 to 8 biological replicates. *P < .05, **P < .01, ****P < .0001 (1-way analysis of variance).

Ablation of the αIIb C490-C545 disulfide bond restricts the distribution of αIIbβ3 to focal adhesions. (A) BHK cells were transfected with WT β3 and either WT or cysteine mutant αIIb and cells sorted for high β3 expression. Expression of β3 in the clones used for the experiments is shown as a bar graph (i) or representative histogram (ii). The bars and errors are mean ± standard error of the mean of 6 biological replicates. (B) Ablation of the αIIb C490-C545 disulfide bond reduces adhesion of cells to immobilized fibrinogen but not poly-D-lysine. The bars and errors are mean ± standard deviation (SD) of 4 independent experiments with 1 or 2 biological replicates. (C) WT or cysteine mutant αIIb-expressing BHK cells were seeded onto a fibrinogen-coated surface in the presence of β3 mAb VI-PL2 and allowed to adhere for 1 hour. After fixation, cells were stained for vinculin (7F9) and nuclei (DAPI), before being imaged using PerkinElmer’s Opera Phenix High-Content Screening System at ×63 magnification in confocal mode. Scale bar, 50 µm. (D) Integrin clusters were quantified in cells seeded onto poly-D-lysine or fibrinogen and expressed per cell (i) or per square micrometers (ii). Discreet integrin clusters in cells adhered to fibrinogen were more abundant when the αIIb C490-C545 disulfide bond is missing. The bars and errors are mean ± SD of 4 to 8 biological replicates. (E) WT or cysteine mutant αIIb-expressing BHK cells were seeded onto a fibrinogen-coated surface in the presence of β3 mAb VI-PL2 and allowed to adhere for 1 hour. After fixation, cells were stained for vinculin (7F9), phosphorylated FAK (pFAK, phosphoY397 FAK mAb), and nuclei (DAPI ), before being imaged by using PerkinElmer’s Opera Phenix high-content screening system at ×63 magnification in confocal mode. Scale bar, 50 µm. (F) F-actin and pFAK intensity in cells seeded on the fibrinogen-coated surface are expressed per µm2. The bars and errors are mean ± SD of 3 to 8 biological replicates. *P < .05, **P < .01, ****P < .0001 (1-way analysis of variance).

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