Figure 6.
Figure 6. Physical and functional association of PKCδ with VASP. (A) Washed human platelets were stimulated for 180 seconds with collagen (30 μg/mL) or unstimulated (vehicle). Reactions were stopped by addition of ice-cold NP-40 lysis buffer, and PKCδ (i) or VASP (ii) was immunoprecipitated and blotted each for PKCδ and VASP as indicated. The arrows indicate the molecular weight of VASP (i) or PKCδ (ii). The immunoblots are representative of 3 independent experiments. (B) Human platelets adherent to a CRP-coated surface were fixed, permeabilized, and immunostained with 1:250 anti-VASP and 1 μg/mL anti-PKCδ antibodies. VASP and PKCδ were then detected using FITC-conjugated anti–rabbit IgG antibody and TRITC-labeled anti–mouse IgG antibody, respectively. Images shown are the distribution of VASP (left column), the distribution of PKCδ (second column), and their superimposition (third column) from 3 independent experiments. The phase-contrast image of the platelet is also shown (right column). The early stage of platelet attachment, the transient filopodial phase, and the late lamellipodial phase are represented on the top, center, and bottom rows, respectively. (C) Whole cell lysates (WCL) of (i) human platelets or (ii) platelets from either wild-type or PKCδ–/– mice were immunoblotted with 1:1000 anti–phospho-VASP (Ser157) and 1:1000 anti–phospho-VASP (Ser239) (top and middle panels, respectively). Bottom panels show the reblot with 1:1000 anti-VASP antibody. Platelets were either nonstimulated or stimulated with CRP (5 μg/mL) or collagen (30 μg/mL) in the absence or presence of rottlerin (5 μM, 15 minutes of preincubation) as indicated. The arrow indicates the molecular weight of VASP. All immunoblots shown are representative of 3 independent experiments. (D) Human platelets were either nonstimulated or stimulated with collagen (30 μg/mL) in the absence or presence of kinase inhibitors (H89, 3 μM; or Gö6976, 1 μM; 15 minutes of preincubation) as indicated. Whole cell lysates (WCL) were immunoblotted with 1:1000 anti–phospho-VASP (Ser157) and reprobed with 1:1000 anti-VASP antibody (top and bottom panels, respectively). The arrow indicates the molecular weight of VASP. All immunoblots shown are representative of 3 independent experiments. (Ei) Human platelets adherent to collagen or (ii) platelets from either wild-type or PKCδ–/– mice adherent to CRP were immunostained with 1:250 anti-VASP antibody and 2 μg/mL TRITC-phalloidin and then with FITC-conjugated anti–rabbit IgG antibody. (i) Distribution of VASP (left column) and F-actin (second column) in human platelets in the absence (top panels) or presence (bottom panels) of rottlerin (5 μM, 15 minutes of preincubation). (ii) Distribution of the same proteins in platelets derived from wild-type (WT, top panels) or PKCδ–/– (bottom panels) mice. The superimposition of VASP and F-actin labeling (third column) and the phase-contrast image (right) are also shown. Images shown are representative of 3 independent experiments.

Physical and functional association of PKCδ with VASP. (A) Washed human platelets were stimulated for 180 seconds with collagen (30 μg/mL) or unstimulated (vehicle). Reactions were stopped by addition of ice-cold NP-40 lysis buffer, and PKCδ (i) or VASP (ii) was immunoprecipitated and blotted each for PKCδ and VASP as indicated. The arrows indicate the molecular weight of VASP (i) or PKCδ (ii). The immunoblots are representative of 3 independent experiments. (B) Human platelets adherent to a CRP-coated surface were fixed, permeabilized, and immunostained with 1:250 anti-VASP and 1 μg/mL anti-PKCδ antibodies. VASP and PKCδ were then detected using FITC-conjugated anti–rabbit IgG antibody and TRITC-labeled anti–mouse IgG antibody, respectively. Images shown are the distribution of VASP (left column), the distribution of PKCδ (second column), and their superimposition (third column) from 3 independent experiments. The phase-contrast image of the platelet is also shown (right column). The early stage of platelet attachment, the transient filopodial phase, and the late lamellipodial phase are represented on the top, center, and bottom rows, respectively. (C) Whole cell lysates (WCL) of (i) human platelets or (ii) platelets from either wild-type or PKCδ–/– mice were immunoblotted with 1:1000 anti–phospho-VASP (Ser157) and 1:1000 anti–phospho-VASP (Ser239) (top and middle panels, respectively). Bottom panels show the reblot with 1:1000 anti-VASP antibody. Platelets were either nonstimulated or stimulated with CRP (5 μg/mL) or collagen (30 μg/mL) in the absence or presence of rottlerin (5 μM, 15 minutes of preincubation) as indicated. The arrow indicates the molecular weight of VASP. All immunoblots shown are representative of 3 independent experiments. (D) Human platelets were either nonstimulated or stimulated with collagen (30 μg/mL) in the absence or presence of kinase inhibitors (H89, 3 μM; or Gö6976, 1 μM; 15 minutes of preincubation) as indicated. Whole cell lysates (WCL) were immunoblotted with 1:1000 anti–phospho-VASP (Ser157) and reprobed with 1:1000 anti-VASP antibody (top and bottom panels, respectively). The arrow indicates the molecular weight of VASP. All immunoblots shown are representative of 3 independent experiments. (Ei) Human platelets adherent to collagen or (ii) platelets from either wild-type or PKCδ–/– mice adherent to CRP were immunostained with 1:250 anti-VASP antibody and 2 μg/mL TRITC-phalloidin and then with FITC-conjugated anti–rabbit IgG antibody. (i) Distribution of VASP (left column) and F-actin (second column) in human platelets in the absence (top panels) or presence (bottom panels) of rottlerin (5 μM, 15 minutes of preincubation). (ii) Distribution of the same proteins in platelets derived from wild-type (WT, top panels) or PKCδ–/– (bottom panels) mice. The superimposition of VASP and F-actin labeling (third column) and the phase-contrast image (right) are also shown. Images shown are representative of 3 independent experiments.

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