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First published online 22 April 2008
doi: 10.1242/jcs.014076

Journal of Cell Science 121, 1641-1648 (2008)
Published by The Company of Biologists 2008
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Analysis of Fyn function in hemostasis and {alpha}IIbβ3-integrin signaling

Kumar B. Reddy*, Dawn M. Smith and Edward F. Plow

Department of Molecular Cardiology and Joseph J. Jacobs Center for Thrombosis & Vascular Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA


Figure 1
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  		Fig. 1. Co-immunoprecipitation of {alpha}IIbβ3 with SFKs from platelets. (A) Equal amounts of total protein in the Triton-X-100-soluble extract of resting platelets or thrombin-treated (aggregated) platelets were subjected to immunoprecipitation using indicated anti-SFK antibodies. The immunoprecipitates were subjected to immunoblot analysis using anti-β3 antibodies. AP3 antibody (lane 2; anti-β3 antibody; positive control) but not control Ig (lane 1; normal mouse serum) immunoprecipitated β3 integrin (bottom panel). (B) Densitometric analysis (n=3) of blots in panel A. (C) Anti-SFK antibodies immunoprecipitated equal amounts of Src and Fyn from resting and thrombin-treated platelets.

 

Figure 2
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  		Fig. 2. Images showing the cellular localization of Fyn, Src and {alpha}IIbβ3 in CHO cells. CHO cells stably expressing the {alpha}IIbβ3 were transfected with the cDNAs encoding either Fyn or Src. 48 hours after transfection, cells were allowed to spread for an additional 24 hours on coverslips coated with fibrinogen. Cells were fixed and subjected to immunohistochemistry using antibodies against Fyn, Src and β3 integrin. Cells were simultaneously stained with fluorescently labeled phalloidin. Nuclei are shown in blue. Scale bars, 20 µm.

 

Figure 3
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  		Fig. 3. Fyn associates with both the full-length and the deletion mutant of {alpha}IIbβ3. (A,B) CHO cells stably expressing either full-length or truncated β3-subunits, together with the full-length {alpha}IIb subunit, were transiently transfected with the cDNAs encoding (A) GFP-Src or (B) GFP-Fyn. 48 hours after transfection, cells were re-plated on fibrinogen-coated plates for 1 hour. Cell extracts were subjected to immunoprecipitation using antibodies against the β3 subunit. The immunoprecipitates were then analyzed for (A) Src or (B) Fyn using anti-GFP antibodies. Expression levels of transfected integrins and GFP fusions are also shown.

 

Figure 4
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  		Fig. 4. Differential binding of SFKs with the GST-β3-spliced variants. (A) Amino acid sequences of β3 variants used in this study. Identical residues between the variants are boxed. (B) Various GST-β3 tails were incubated with the platelet lysates. The captured proteins were subjected to SDS-PAGE followed by western blot analyses using indicated antibodies. The protein loading is shown in the Coomassie stain.

 

Figure 5
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  		Fig. 5. Identification of the Fyn-binding region within the β3-cytoplasmic domain. (A) Amino acid sequences of β3-deletion mutants used in this study. (B) The different GST-β3-deletion mutants were incubated with the platelet lysates. Proteins were subjected to SDS-PAGE followed by western blot analyses using anti-Fyn antibodies. The protein loading is shown in the Coomassie stain.

 

Figure 6
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  		Fig. 6. Fyn–/– mice exhibit prolonged second bleeding times. Tail-bleeding times were measured in Fyn–/– and the corresponding wild-type (WT) mice (n=11) as described in Materials and Methods. Second bleeding was allowed for 5 minutes, at which point the experiment was stopped and tails were pressured to stop bleeding. The mean second bleeding time in Fyn–/– and WT mice were 3.61 minutes and 1.047 minutes, respectively; P=0002.

 

Figure 7
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  		Fig. 7. Spreading of platelets on fibrinogen-coated surface. (A) Washed platelets from wild-type (WT) and Fyn–/– mice were plated onto fibrinogen-coated coverslips. Pooled platelets from three to four mice were used for these studies. Platelet numbers were 106 cells per well in six-well plates. Cells were allowed to adhere for 30 minutes, fixed, stained with fluorescently labeled phalloidin and then images were captured. Scale bar, 10 µm. (B) The cells from six different fields (a total of 200-300 cells) were counted to determine the percentage spread cells. Data from an independent experiment are given in the bottom panel.

 

Figure 8
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  		Fig. 8. Transient expression of Fyn restores cell spreading in CHO cells that express the truncated form of β3-integrin. (A) CHO cells expressing the deletion mutant {alpha}IIbβ3{Delta}754 were transfected with the cDNAs encoding either Fyn (top panels) or Src (bottom panels). 48 hours after transfection, cells were allowed to spread for 30 minutes in serum-free medium on coverslips coated with fibrinogen. Cells were fixed and stained with TRITC-labeled phalloidin. Cells transfected with Fyn and Src were identified by the GFP fluorescence (indicated with arrows). (B) Cells were transfected with cDNA encoding a kinase-dead version of Fyn (FynK299M) and then simultaneously stained using anti-Fyn antibodies and phalloidin. Scale bars, 20 µm.

 

Figure 9
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  		Fig. 9. Transient expression of kinase-dead version of Fyn fails to localize to focal adhesions. (A,B) CHO-{alpha}IIbβ3 were transfected with the cDNAs coding for (A) a kinase-dead version of Fyn (FynK299M) or (B) a constitutively active form of Fyn (Fyn{Delta}525). Transfected cells were allowed to spread for 24 hours on coverslips coated with fibrinogen. Cells were fixed and stained for Fyn (red), actin (green) and nuclei (blue). Scale bar, 20 µm.

 

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