Figure 5.
Figure 5. Fluorescence micrographs showing the localization of WAVEs, WASP, cortactin, Arp3, and F-actin in human platelets. (A) Platelets were allowed to attach and spread on fibrinogen-coated (0.1 mg/mL) or fibronectin-coated (20 μg/mL) or glass coverslips and were fixed and stained for WAVEs with specific affinity-purified antibodies. Platelets spread on uncoated glass coverslips were stained for F-actin, WASP, Arp3, and cortactin as indicated. Before use, coated coverslips were blocked with bovine serum albumin (1% in phosphate buffered saline) for 1 hour at 37°C. (B) WAVE2 is observed in the rim of the lamellipodia following permeabilization and fixation of spread platelets and stained with anti-WAVE2. The presence of WAVE2 was examined as in panel A, except that spread platelets were fixed in 4% paraformaldehyde in PHEM buffer (described in “Materials and methods”) containing a 1/50 dilution of phalloidin with 0.25% Triton for 20 minutes at room temperature. (C) The presence of Abi-1. Platelet whole-cell lysate was subjected to SDS-PAGE, followed by immunoblotting using an anti–Abi-1 monoclonal antibody (4E2). (D) Colocalization of WAVE2 and Abi-1. Fixation of spread platelets was done as in panel B, except that the sample was stained with both anti-WAVE2 and anti–Abi-1 (4E2). (E) Association of WAVE/Scars and Abi-1. Whole-cell lysates from resting platelets were subjected to immunoprecipitation using either anti–Abi-1 (1G9) or control IgG (C). The precipitates were equally divided into 4 aliquots; SDS-PAGE and immunoblotting were performed by using anti-WAVE1 (left), anti-WAVE2 (middle), anti-WAVE3 (right), and anti–Abi-1(1G9; bottom right). The blotting was performed on the same membrane. HC and LC indicate heavy and light chains of IgG. The relative positions of Wave/Scars are indicated by arrows. (F) WIP, Abi-1, WASP, Sra-1, and WASP are precipitated by GST-SH3 of abl but not GST. For WIP, Abi-1, and WASP, platelet proteins were precipitated by GST-SH3 of abl. The precipitates were subjected to SDS-PAGE, followed by immunoblotting by using the antibodies indicated. For Sra-1 and Nap-1, the FLAG-tagged proteins were expressed in Cos7 cells. Equal amount of the cell lysates were incubated with GST-SH3 of abl or GST alone. The precipitates were subjected to SDS-PAGE, followed by immunoblotting by using an anti-FLAG monoclonal antibody.

Fluorescence micrographs showing the localization of WAVEs, WASP, cortactin, Arp3, and F-actin in human platelets. (A) Platelets were allowed to attach and spread on fibrinogen-coated (0.1 mg/mL) or fibronectin-coated (20 μg/mL) or glass coverslips and were fixed and stained for WAVEs with specific affinity-purified antibodies. Platelets spread on uncoated glass coverslips were stained for F-actin, WASP, Arp3, and cortactin as indicated. Before use, coated coverslips were blocked with bovine serum albumin (1% in phosphate buffered saline) for 1 hour at 37°C. (B) WAVE2 is observed in the rim of the lamellipodia following permeabilization and fixation of spread platelets and stained with anti-WAVE2. The presence of WAVE2 was examined as in panel A, except that spread platelets were fixed in 4% paraformaldehyde in PHEM buffer (described in “Materials and methods”) containing a 1/50 dilution of phalloidin with 0.25% Triton for 20 minutes at room temperature. (C) The presence of Abi-1. Platelet whole-cell lysate was subjected to SDS-PAGE, followed by immunoblotting using an anti–Abi-1 monoclonal antibody (4E2). (D) Colocalization of WAVE2 and Abi-1. Fixation of spread platelets was done as in panel B, except that the sample was stained with both anti-WAVE2 and anti–Abi-1 (4E2). (E) Association of WAVE/Scars and Abi-1. Whole-cell lysates from resting platelets were subjected to immunoprecipitation using either anti–Abi-1 (1G9) or control IgG (C). The precipitates were equally divided into 4 aliquots; SDS-PAGE and immunoblotting were performed by using anti-WAVE1 (left), anti-WAVE2 (middle), anti-WAVE3 (right), and anti–Abi-1(1G9; bottom right). The blotting was performed on the same membrane. HC and LC indicate heavy and light chains of IgG. The relative positions of Wave/Scars are indicated by arrows. (F) WIP, Abi-1, WASP, Sra-1, and WASP are precipitated by GST-SH3 of abl but not GST. For WIP, Abi-1, and WASP, platelet proteins were precipitated by GST-SH3 of abl. The precipitates were subjected to SDS-PAGE, followed by immunoblotting by using the antibodies indicated. For Sra-1 and Nap-1, the FLAG-tagged proteins were expressed in Cos7 cells. Equal amount of the cell lysates were incubated with GST-SH3 of abl or GST alone. The precipitates were subjected to SDS-PAGE, followed by immunoblotting by using an anti-FLAG monoclonal antibody.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal