Analysis of Fyn function in hemostasis and IIbβ3-integrin signaling

JCS014076 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Expression of various Src-family kinases (SFKs) in platelets from WT and Fyn^−/− mice. Equal amounts of total protein from the triton-soluble extract of WT and Fyn^−/− mice were subjected to immunoblot analyses using the indicated antibodies. Anti-cortactin antibodies were used as a loading control.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Immunoblot analysis of Src and β3 integrin in various platelet fractions. Washed human platelets were not stimulated or thrombin stimulated. Equal amounts of total proteins from triton-soluble and triton-insoluble fractions were subjected to immunoblot analyses using the indicated antibodies (top panels). There was approximately ten times more protein in the triton-soluble compared with triton-insoluble fractions. Equal amounts of proteins from control and thrombin-treated platelets were subjected to immunoblot analysis using antibodies that recognize a calpain cleaved form of β3 integrin (residue 754) (Du et al., 1995).

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. Images show the localization of FAK and Fyn. (A) CHO cells stably expressing αIIbβ3 integrin were transfected with the cDNA encoding Src. 48 hours after transfection, the cells were allowed to spread for an additional 24 hours on coverslips coated with fibrinogen. Cells were stained with anti-FAK antibodies. GFP-Src-expressing cells are indicated with asterisks. (B) 3A-sub E cells (human placental cell line; ATCC: CRL-1584) were grown on fibrinogen-coated coverslips and subjected to immunohistochemistry using antibodies to Fyn. Arrows indicate the focal adhesions. Bar: 10 µm.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. Images show the presence of β3 inegrins on 3A-sub E cells. 3A-sub E cells were grown on fibrinogen-coated coverslips and subjected to immunohistochemistry using either anti-β1- or anti-β3-integrin antibodies. As shown in the left panel, β3 integrin, but not β1 integrinis, is readily detected on these cells. Bar: 10 µm.

• Supplemental Figure S5 (Adobe PDF) -

  Fig. S5. Platelet-aggregation studies. Gel-filtered platelets (∼3×10⁷ cells/ml) from WT or Fyn^−/− mice were subjected to agonist-induced aggregation using thrombin (0.1 U), collagen (5 µg/ml) and ADP (50 µM) as stimuli.

• Supplemental Figure S6 (Adobe PDF) -

  Fig. S6. Spreading of platelets on collagen coated surface. Washed platelets from WT and Fyn^−/− mice were plated onto collagen-coated coverslips (BD Biosciences). 1×10⁶ cells each were plated in six-well plates. The cells were allowed to adhere for 30 minutes, fixed, stained with fluorescently labeled phalloidin and images were taken. Bar: 10 µm. The cells from four different fields were counted to determine the percentage of spread cells.

• Supplemental Figure S7 (Adobe PDF) -

  Fig. S7. Quantification of platelets spreading after various treatments. Washed platelets form WT or Fyn^−/− mice were plated onto fibrinogen-coated coverslips; 1×10⁶ cells per well were plated six-well plates. Some of them were treated with 20 µM ADP with or without 10 U of apyrase. The cells were allowed to adhere for 60 minutes, fixed, stained with fluorescently labeled phalloidin, and images were taken. The cells from six different fields were counted to determine the percentage of spread cells.