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1.
Figure 1

Figure 1. Murine and human platelets express different members of the paxillin family. From: Paxillin family members function as Csk-binding proteins that regulate Lyn activity in human and murine platelets.

(A) Hic-5 in human platelet lysates was detected by immunoblotting (IB) with an anti-human Hic-5 antibody that does not cross-react with Hic-5 in murine platelet lysates. (B) Hic-5 (55 kDa) in human platelet lysates, and paxillin (68 kDa), Hic-5 (53 kDa) and leupaxin (45 kDa) in murine platelet lysates, were detected with an anti-paxillin antibody that cross-reacts with Hic-5 and leupaxin.

Vipul B. Rathore, et al. Biochem J. 2007 April 15;403(Pt 2):275-281.
2.
Figure 5

Figure 5. SFK Lyn, but not Src or Fyn, is constitutively associated with paxillin family members in platelets. From: Paxillin family members function as Csk-binding proteins that regulate Lyn activity in human and murine platelets.

Washed human (A) and murine (B) platelets were stirred without or with CRP for 3 min. Lysates were prepared and subjected to immunoprecipitation with an anti-paxillin antibody that cross-reacts with Hic-5 and leupaxin. Lysates and immunoprecipitates (IP) were immunoblotted (IB) with antibodies specific for p60Src (i, a), p59Fyn (ii, a) and p53/56Lyn (iii, a). Parallel immunoprecipitates were immunoblotted with anti-human Hic-5 (A: i,b; ii,b; and iii,b) or with an anti-paxillin antibody that cross-reacts with Hic-5 and leupaxin (B: i,b; ii,b; and iii,b) to show equal antigen loading.

Vipul B. Rathore, et al. Biochem J. 2007 April 15;403(Pt 2):275-281.
3.
Figure 2

Figure 2. Paxillin family members in platelets were tyrosine phosphorylated in aggregated platelets. From: Paxillin family members function as Csk-binding proteins that regulate Lyn activity in human and murine platelets.

(A) Hic-5 was tyrosine phosphorylated in aggregated human platelets. (B) Paxillin and Hic-5, but not leupaxin, become tyrosine phosphorylated in aggregated murine platelets. Washed human and murine platelets were stirred without or with CRP for 3 min. Lysates were prepared and subjected to immunoprecipitation with an antibody raised against paxillin that also cross reacts well with Hic-5 and leupaxin. As a control, lysates from platelets stirred without CRP were subjected to immunoprecipitation with normal mouse immnunoglobulin (NMIg). Immunoprecipitates (IP) were immunoblotted (IB) with a phosphotyrosine-specific antibody (PY) as described in the Materials and methods section.

Vipul B. Rathore, et al. Biochem J. 2007 April 15;403(Pt 2):275-281.
4.
Scheme 1

Scheme 1. A model of regulation of Lyn activity by Csk binding to members of the paxillin family in aggregated human and murine platelets. From: Paxillin family members function as Csk-binding proteins that regulate Lyn activity in human and murine platelets.

(A) Lyn, via its SH3 domain, is bound constitutively to the polyproline region of paxillin or Hic-5, both of which are unphosphorylated in resting platelets. (B) Paxillin and Hic-5 become tyrosine phosphorylated during platelet aggregation, leading to recruitment of Csk from the cytosol and juxtaposition of the kinase domain of Csk with the C-terminal inhibitory tyrosine (Y507) of Lyn. (C) Csk phosphorylates of Tyr507 of Lyn resulting in an intramolecular interaction between phospho-Tyr507 and the SH2 domain of Lyn that inhibits Lyn activity.

Vipul B. Rathore, et al. Biochem J. 2007 April 15;403(Pt 2):275-281.
5.
Figure 6

Figure 6. Csk-mediated inhibition of paxillin/Hic-5-associated Lyn requires αIIbβ3 engagement in human (A) and murine (B) platelets. From: Paxillin family members function as Csk-binding proteins that regulate Lyn activity in human and murine platelets.

Platelets were stimulated with CRP in the presence (activated platelets) or absence (aggregated platelets) of RGDW and EDTA as described in the Materials and methods section. Lysates were prepared and immunoprecipitated with an anti-paxillin antibody that cross-reacts with Hic-5 and leupaxin. Immunoprecipitates (IP) were immunoblotted (IB) with antibodies specific for the phosphorylated form of the C-terminal inhibitory tyrosine residue of Lyn (pLyn507), Lyn, Csk, phosphotyrosine (PY) and either human Hic-5 (A) or paxillin/Hic-5/leupaxin (B).

Vipul B. Rathore, et al. Biochem J. 2007 April 15;403(Pt 2):275-281.
6.
Figure 7

Figure 7. Csk-binding functions of paxillin family members are differentially activated in human and murine platelets. From: Paxillin family members function as Csk-binding proteins that regulate Lyn activity in human and murine platelets.

Binding of fibrinogen to activated αIIbβ3 does not induce tyrosine phosphorylation of, or recruitment of Csk to, Hic-5 in human platelets (A), but is sufficient to induce tyrosine phosphorylation of, and recruitment of Csk to, paxillin and Hic-5 in murine platelets (B) and (C). Platelets were incubated under resting conditions (R), in the presence of 250 μg/ml fibrinogen alone (F), 0.5 mM MnCl2 alone (M), or fibrinogen and MnCl2 together (F + M). The latter condition supports binding of added fibrinogen to αIIbβ3 integrins that are directly activated by MnCl2. Platelet lysates were prepared and subjected to immunoprecipitation (IP) with an anti-paxillin antibody that cross-reacts with Hic-5 and leupaxin. Immunoprecipitates were separated by SDS/PAGE and immunoblotted (IB) with antibodies specific for phosphotyrosine (PY), Csk and either human Hic-5 (A) or paxillin/Hic-5/leupaxin (B).

Vipul B. Rathore, et al. Biochem J. 2007 April 15;403(Pt 2):275-281.
7.
Figure 4

Figure 4. Hic-5 in human platelets and paxillin in murine platelets function as Csk-binding proteins. From: Paxillin family members function as Csk-binding proteins that regulate Lyn activity in human and murine platelets.

(A) Hic-5 co-immunoprecipitates with the SH2-domain of Csk from CRP-aggregated but not resting human platelets. (B) Paxillin predominantly, and Hic-5 weakly, co-immunoprecipitate with the SH2-domain of Csk from CRP-aggregated but not resting murine platelets. Washed platelets were stirred without or with CRP for 3 min, after which platelet lysates were prepared. Lysates were precleared by incubating with GST conjugated to glutathione–Sepharose beads to remove non-specific binding proteins and the GST–Csk-SH2 pull-down was performed as described in the Materials and methods section. Paxillin family members associated with GST–Csk-SH2 fusion protein (left-hand panels) and those present in platelet lysates (right-hand panels) were analysed by immunoblotting (IB) with anti-human Hic-5 (A) or with an anti-paxillin antibody that cross-reacts with Hic-5 and leupaxin (B).

Vipul B. Rathore, et al. Biochem J. 2007 April 15;403(Pt 2):275-281.
8.
Figure 3

Figure 3. Csk co-immunoprecipitates with tyrosine-phosphorylated Hic-5 from CRP-stimulated human platelet lysates and with tyrosine-phosphorylated paxillin/Hic-5 from murine platelet lysates. From: Paxillin family members function as Csk-binding proteins that regulate Lyn activity in human and murine platelets.

Washed human (A and B) and murine (C and D) platelets were stirred without or with CRP for 3 min. Lysates were prepared and subjected to immunoprecipitation (IP) with an anti-paxillin antibody that cross-reacts with Hic-5 and leupaxin. Immunoprecipitates were immunoblotted (IB) with anti-Csk (A and C, top panels), anti-phosphotyrosine (PY; A and C, middle panels) and either anti-Hic-5 (A, bottom panel) or anti-paxillin/Hic-5/leupaxin (C, bottom panel). The amount of Csk that co-immunoprecipitated with paxillin and/or Hic-5 was determined by densitometry and is reported as the mean band intensity±S.D. observed in three and two independent experiments performed from human (B) and murine (D) platelets respectively. *, A paired t test was used to determine statistical significance. Note that CRP-induced aggregation significantly enhanced association of Csk with Hic-5 in human platelets (P<0.03) and with paxillin/Hic-5 in murine platelets (P<0.04).

Vipul B. Rathore, et al. Biochem J. 2007 April 15;403(Pt 2):275-281.

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