Figure 4.
Figure 4. Filamin and GPIb coimmunoprecipitate with SHIP-2. (A) The Triton X-100–soluble fraction of resting, thrombin-stimulated (1 U/mL, final concentration for 5 minutes), or VWF-stimulated platelets (10 μg/mL, and botrocetin, 3 μg/mL; final concentration for 5 minutes) was isolated. The Triton X-100–soluble fraction was immunoprecipitated with either preimmune (Pre I) sera or SHIP-2 antibodies and immunoblotted with filamin or GPIb antibodies. (B) The Triton X-100–soluble fraction of resting and thrombin-stimulated platelets was isolated as described in panel A, immunoprecipitated with either nonimmune (Non I) sera or filamin antibodies and immunoblotted with either affinity-purified SHIP-2 antibodies or GPIb antibodies. (C) The Triton X-100–soluble fraction of resting and thrombin-stimulated platelets was isolated as described in panel A, immunoprecipitated with either nonimmune (Non I) sera or GPIb antibodies and immunoblotted with affinity-purified SHIP-2 antibodies or filamin antibodies. (D) The Triton X-100–soluble fraction of resting, thrombin-stimulated (1 U/mL, final concentration for 5 minutes), or VWF-stimulated platelets (10 μg/mL, and botrocetin, 3 μg/mL; final concentration for 5 minutes) was isolated. The Triton X-100–soluble fraction was immunoprecipitated with either nonimmune (Non I) sera, SHIP-2, filamin, or GPIb antibodies and immunoblotted with the immunoprecipitating antibody. (E) Triton X-100–soluble fractions were isolated from untreated platelets and immunoprecipitated with either nonimmune (Non I) sera, affinity-purified SHIP-2 antibodies, filamin antibodies, or GPIb antibodies. The Triton X-100–soluble fraction was also isolated from thrombin-stimulated (1 U/mL, final concentration for 5 minutes) platelets and immunoprecipitated with either nonimmune (Non I) sera or affinity-purified SHIP-2 antibodies. Immunoprecipitates captured on protein-A–Sepharose were subjected to PtdIns(3,4,5)P3 5-phosphatase assays and the lipid products of the enzyme assay were examined by TLC. The migration of the phospholipids was compared with known standards PtdIns(3,4,5)P3 and PtdIns(3,4)P2.

Filamin and GPIb coimmunoprecipitate with SHIP-2. (A) The Triton X-100–soluble fraction of resting, thrombin-stimulated (1 U/mL, final concentration for 5 minutes), or VWF-stimulated platelets (10 μg/mL, and botrocetin, 3 μg/mL; final concentration for 5 minutes) was isolated. The Triton X-100–soluble fraction was immunoprecipitated with either preimmune (Pre I) sera or SHIP-2 antibodies and immunoblotted with filamin or GPIb antibodies. (B) The Triton X-100–soluble fraction of resting and thrombin-stimulated platelets was isolated as described in panel A, immunoprecipitated with either nonimmune (Non I) sera or filamin antibodies and immunoblotted with either affinity-purified SHIP-2 antibodies or GPIb antibodies. (C) The Triton X-100–soluble fraction of resting and thrombin-stimulated platelets was isolated as described in panel A, immunoprecipitated with either nonimmune (Non I) sera or GPIb antibodies and immunoblotted with affinity-purified SHIP-2 antibodies or filamin antibodies. (D) The Triton X-100–soluble fraction of resting, thrombin-stimulated (1 U/mL, final concentration for 5 minutes), or VWF-stimulated platelets (10 μg/mL, and botrocetin, 3 μg/mL; final concentration for 5 minutes) was isolated. The Triton X-100–soluble fraction was immunoprecipitated with either nonimmune (Non I) sera, SHIP-2, filamin, or GPIb antibodies and immunoblotted with the immunoprecipitating antibody. (E) Triton X-100–soluble fractions were isolated from untreated platelets and immunoprecipitated with either nonimmune (Non I) sera, affinity-purified SHIP-2 antibodies, filamin antibodies, or GPIb antibodies. The Triton X-100–soluble fraction was also isolated from thrombin-stimulated (1 U/mL, final concentration for 5 minutes) platelets and immunoprecipitated with either nonimmune (Non I) sera or affinity-purified SHIP-2 antibodies. Immunoprecipitates captured on protein-A–Sepharose were subjected to PtdIns(3,4,5)P3 5-phosphatase assays and the lipid products of the enzyme assay were examined by TLC. The migration of the phospholipids was compared with known standards PtdIns(3,4,5)P3 and PtdIns(3,4)P2.

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