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. 2015 Jan 21;290(10):6226–6242. doi: 10.1074/jbc.M114.634436

FIGURE 9.

FIGURE 9.

Kindlin-3 phosphomutant (S482A/T484A (S482T484/AA)) blunts integrin-induced cell spreading and soluble ligand binding. A, HEL cells were transiently transfected with plasmids encoding EGFP alone and EGFP-kindlin-3 constructs. EGFP expression levels in HEL cells were determined by flow cytometry; transfection efficiency was 70–80%. Transfected HEL cells were treated with PMA and allowed to adhere to fibrinogen coated coverslips, and cell spreading was measured after 30 min. The adherent cells were fixed and stained with Alexa 568 phalloidin. The areas of cells were measured using ImageJ software, and 300 cells were quantified in each experiment. The error bars represent means ± S.D. of three independent experiments (*, p < 0.001). B, plasmids encoding EGFP alone or EGFP-kindlin-3 constructs were transiently transfected to HEL cells. HEL cells were stimulated with 800 nm PMA, and the transfected cells were stained with PAC-1 to assess αIIbβ3 activation. Flow cytometry was used to measure the MFI of PAC-1 binding. Integrin expression of transfected cells was measured in parallel with 2G12 antibody that binds αIIbβ3 integrin in an activation-independent manner. The error bars represent means ± S.D. of three independent experiments (*, p < 0.001). C, plasmids encoding EGFP alone or EGFP-kindlin-3 constructs were transiently transfected to K562 αIIbβ3 cells. EGFP expression levels were determined by flow cytometry with a transfection efficiency of 70–80%. The transfected cells were stimulated with 800 nm PMA and incubated with 20 μg/ml human fibrinogen, and the flow cytometry was used to measure the MFI of fibrinogen binding. The error bars represent means ± S.E. of three independent experiments (*, p < 0.001). D, mouse RAW 264.7 cells were transiently transfected with plasmids encoding EGFP alone or EGFP-kindlin-3 constructs. The transfected cells were stained with 9EG7 monoclonal antibody to assess β1 integrin activation. Flow cytometry was used to measure the MFI of 9EG7 binding. The error bars represent means ± S.E. of three independent experiments (*, p < 0.001).