|
|
Blood, Vol. 108, Issue 13, 4035-4044, December 15, 2006
PKC regulates collagen-induced platelet aggregation through inhibition of VASP-mediated filopodia formation
Blood Pula et al.
108: 4035
Supplemental materials for: Pula et al, Vol 108, Issue 13, 4035-4044
Files in this Data Supplement:
- Figure S1. NSC23766 inhibits Rac1 activation (JPG, 50.5 KB)
-
The activity of NSC23766 was investigated by Rac1 pull-down using GST-tagged PAK-PBD protein beads. Washed human platelets were either not stimulated or stimulated with collagen (30 µg/mL) in the absence or presence of NSC23766 (200 µM, 15 minutes of preincubation). Platelet lysis and pull-downs were performed as described in “Materials and methods” in the main article, and the amount of GTP-loaded Rac1 was assessed by immunoblot. As a positive control, platelet extracts were treated with GTPγS (200 µM), while platelet extracts treated with GDP (1 mM) were used as negative control. Images shown are representative of 3 independent experiments.
- Figure S2. PKCδ does not regulate PKA or PKG activity (JPG, 94 KB)
-
(Ai) Platelets were stimulated with collagen (30 µg/mL) in the absence or presence of rottlerin (5 µM, 15 minutes preincubation) as indicated. (ii) As a positive control, whole cell lysates from human platelets were either untreated (basal) or treated with forskolin (10 µM, 15 minutes) to activate adenylate cyclase and thereby PKA. Whole cell lysates (WCLs) were immunoblotted with 1:1000 anti–phospho-Ser/Thr PKA substrate antibody, which recognizes PKA kinase consensus sequences in substrate proteins when phosphorylated. (Bi) Platelets were stimulated with collagen (30 µg/mL) in the absence or presence of rottlerin (5 µM, 15 minutes of preincubation) as indicated. (ii) As a positive control, whole cell lysates from human platelets were either untreated (basal) or treated with 8-pCPT-cGMP (200 µM, 15 minutes). WCLs were immunoblotted with 1:1000 PhosphoDetect anti–protein kinase G (PKG) substrate antibody, which recognizes PKG kinase consensus sequences in substrate proteins when phosphorylated. All immunoblots shown are representative of 3 independent experiments.
- Figure S3. PKCδ does not regulate the activity of classical PKC isoforms (JPG, 90.1 KB)
-
(A) Human and (B) either wild-type (WT) or PKC –/– mouse platelets were either unstimulated or stimulated with collagen (30 µg/mL), and in the case of human platelets rottlerin was added where indicated (5 µM, 15 minutes of preincubation). WCLs were generated, and protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with 1:1000 anti–phospho-Ser PKC substrate antibody, which recognizes classical PKC kinase consensus sequences in substrate proteins when phosphorylated. All immunoblots shown are representative of 3 independent experiments.
- Video S1. Transient filopodia generation in human platelets adherent to collagen fibers (MOV, 1.24 MB)
-
Washed human platelets were added to collagen-coated coverslips and allowed to settle on the surface and adhere to collagen fibers under static conditions. Collagen fibers may be seen as hairlike strands across the field of view. Platelets were visualized by differential interference contrast (DIC) microscopy, and images were captured once every 10 seconds and displayed in the video at a 120× accelerated rate. It may be seen that upon adhesion to the collagen fiber in the field of view, platelets undergo a rapid change in shape involving a transient generation of filopodia that is rapidly replaced by persistent lamellipodia in the fully spread platelet. This video is representative of 5 independent experiments.
- Video S2. Sustained filopodia generation in human platelets pretreated with rottlerin adherent to collagen fibers (MOV, 3.95 MB)
-
Washed human platelets, pretreated for 5 minutes with rottlerin (5 µM), were added to collagen-coated coverslips and allowed to settle on the surface and adhere to collagen fibers under static conditions. Collagen fibers may be seen as hairlike strands across the field of view. Platelets were visualized by DIC microscopy, and images were captured once every 10 seconds and displayed in the video at a 120× accelerated rate. It may be seen that upon adhesion to the collagen fiber in the field of view, platelets undergo a rapid change in shape. In contrast to control platelets shown in Video S1, where filopodia generation is a transient event, it may be seen that in the presence of rottlerin (5 µM) filopodia formation is a sustained event. This is superimposed upon a persistent lamellipodia formation in the fully spread platelet. This video is representative of 5 independent experiments.
- Video S3. Transient filopodia generation in wild-type mouse platelets adherent to collagen fibers (MOV, 1.86 MB)
-
Washed wild-type mouse platelets were added to collagen-coated coverslips and allowed to settle on the surface and adhere to collagen fibers under static conditions. Collagen fibers may be seen as hairlike strands across the field of view. Platelets were visualized by DIC microscopy, and images were captured once every 10 seconds and displayed in the video at a 120× accelerated rate. It may be seen that, as for human platelets, upon adhesion to the collagen fiber in the field of view, platelets undergo a rapid change in shape involving a transient generation of filopodia that is rapidly replaced by persistent lamellipodia in the fully spread platelet. This video is representative of 5 independent experiments.
- Video S4. Sustained filopodia generation in PKCδ–/– mouse platelets adherent to collagen fibers (MOV, 2.13 MB)
-
Washed PKC–/– mouse platelets were added to collagen-coated coverslips and allowed to settle on the surface and adhere to collagen fibers under static conditions. Collagen fibers may be seen as hairlike strands across the field of view. Platelets were visualized by DIC microscopy, and images were captured once every 10 seconds and displayed in the video at a 120× accelerated rate. It may be seen that upon adhesion to the collagen fiber in the field of view, platelets undergo a rapid change in shape. In contrast to wild-type platelets shown in Video S3, where filopodia generation is a transient event, it may be seen that in the absence of PKC filopodia formation is a sustained event. This is superimposed upon a persistent lamellipodia formation in the fully spread platelet. This video is representative of 5 independent experiments.
|
|
