Journal: PLoS ONE

Article Title: The LDL Receptor-Related Protein 1 (LRP1) Regulates the PDGF Signaling Pathway by Binding the Protein Phosphatase SHP-2 and Modulating SHP-2- Mediated PDGF Signaling Events

doi: 10.1371/journal.pone.0070432

Figure Lengend Snippet: (a) Cell lysates from NRK fibroblasts were incubated with phosphorylated GST:LRP1-ICD ( lane 1 ), unphosphorylated GST:LRP1-ICD ( lane 3 ), GST and src ( lane 2 ) or GST alone ( lane 4 ) all bound to Glutathione-Sepharose. Following incubation and washing, eluted proteins were separated by SDS-PAGE and analyzed by immunoblot analysis for SHP-2 ( upper panel ), for tyrosine phosphorylation ( middle panel ) and for total protein by Ponseau stain ( lower panel ). (b) Increasing concentrations of phosphorylated (circles) or unphosporylated (squares) GST:LRP1-ICD were incubated with microtiter wells coated with SHP-2 (closed symbols) or BSA (open symbols). Bound GST:LRP1-ICD was detected with anti-GST antibodies. Curve shows the best fit to a single class of sites using non-linear regression analysis. *, absorbance values for pLRP1-ICD are significantly different from those of LRP1-ICD (p<0.0001, Students t test) (c) SPR analysis confirms binding of SHP-2 (1 µM) to immobilized phosphorylated GST:LRP1-ICD but not to immobilized unphosphorylated GST:LRP1-ICD.

Article Snippet: Binding of GST:LRP1-ICD to recombinant human SHP-2 (R&D Systems, 1894-SH-100) was measured using a BIA 3000 optical biosensor (BIAcore AB, Uppsala, Sweden).

Techniques: Incubation, SDS Page, Western Blot, Staining, Binding Assay

Journal: PLoS ONE

Article Title: The LDL Receptor-Related Protein 1 (LRP1) Regulates the PDGF Signaling Pathway by Binding the Protein Phosphatase SHP-2 and Modulating SHP-2- Mediated PDGF Signaling Events

doi: 10.1371/journal.pone.0070432

Figure Lengend Snippet: Increasing concentrations of phosphorylated (closed squares) or unphosphorylated PDGFRβ KD (closed circles) were incubated with microtiter wells coated with SHP-2. Bound PDGFRβ KD was detected using an anti-PDGFRβ antibody. As a control, the binding of phosphorylated PDGFRβ KD to BSA (open triangles) was also measured. Curves show the best fit to a single class of sites using non-linear regression analysis. The binding of pPDGFRβ to SHP-2 is significantly different from the binding of PDGFRβ to SHP-2 (p = 0.0002, Students t test).

Article Snippet: Binding of GST:LRP1-ICD to recombinant human SHP-2 (R&D Systems, 1894-SH-100) was measured using a BIA 3000 optical biosensor (BIAcore AB, Uppsala, Sweden).

Techniques: Incubation, Binding Assay

Journal: PLoS ONE

Article Title: The LDL Receptor-Related Protein 1 (LRP1) Regulates the PDGF Signaling Pathway by Binding the Protein Phosphatase SHP-2 and Modulating SHP-2- Mediated PDGF Signaling Events

doi: 10.1371/journal.pone.0070432

Figure Lengend Snippet: (a,b) Increasing concentrations of GST:pLRP1-ICD were incubated with microtiter wells coated with SHP-2 in the presence of 0, 20, 60 and 180 nM pPDGFRβ KD (a) or sPDGFr (b). Bound GST:pLRP1-ICD was detected with anti-GST antibody. Curves in (a) show best fits to a single class of sites using non-linear regression analysis. The K D values at each concentration are significantly different (p = 0.0007, Students t test). (c) Percent binding, normalized to 0 nM pPDGFRβ, of GST:pLRP1-ICD (100 nM) binding to SHP-2 in the presence of 20, 60 and 180 nM pPDGFRβ KD. (*, p = 0.0024; **p<0.0001, Students t test).

Article Snippet: Binding of GST:LRP1-ICD to recombinant human SHP-2 (R&D Systems, 1894-SH-100) was measured using a BIA 3000 optical biosensor (BIAcore AB, Uppsala, Sweden).

Techniques: Incubation, Concentration Assay, Binding Assay

Journal: PLoS ONE

Article Title: The LDL Receptor-Related Protein 1 (LRP1) Regulates the PDGF Signaling Pathway by Binding the Protein Phosphatase SHP-2 and Modulating SHP-2- Mediated PDGF Signaling Events

doi: 10.1371/journal.pone.0070432

Figure Lengend Snippet: (a) Purified PDGFRβ KD was incubated with recombinant human SHP-2 in the presence or absence of ATP and phosphatase inhibitors. Proteins were separated by SDS-PAGE and analyzed by immunoblot analysis for phosphotyrosine ( upper panel ). Total proteins are shown in the lower panel. As a control, SHP-1, a structurally similar phosphatase to SHP-2, was also included (lane 5). (b) The catalytic activity of SHP-2 was assessed using a malachite green phosphatase assay kit using Src pY529 (TSTEPQ-pY-QPGENL) as the substrate. Recombinant SHP-2 was incubated with pPDGFR KD or sPDGFRr, GST:LRP1-ICD, in the presence ATP and MgCl 2 . Liberated phosphate complexed with malachite green was measured at 620 nm (*p<0.0001 one way ANOVA5; *p<0.05 Tukey’s multiple comparison post hoc test).

Article Snippet: Binding of GST:LRP1-ICD to recombinant human SHP-2 (R&D Systems, 1894-SH-100) was measured using a BIA 3000 optical biosensor (BIAcore AB, Uppsala, Sweden).

Techniques: Purification, Incubation, Recombinant, SDS Page, Western Blot, Activity Assay, Phosphatase Assay, Comparison

Journal: PLoS ONE

Article Title: The LDL Receptor-Related Protein 1 (LRP1) Regulates the PDGF Signaling Pathway by Binding the Protein Phosphatase SHP-2 and Modulating SHP-2- Mediated PDGF Signaling Events

doi: 10.1371/journal.pone.0070432

Figure Lengend Snippet: (a) MOVAS cells were serum-starved and incubated with or without dynasore (100 µM) at 37°C for 30 min. Cells were then incubated with or without PDGF-B (30 ng/ml) for 2 and 10 min at 37°C. Cells were lysed and proteins separated by SDS-PAGE and analyzed by immunoblotting using the indicated antibodies. (b) Levels of phosphorylated SHP-2, normalized to total SHP-2 amounts in untreated (control) and dynasore treated cells are plotted.

Article Snippet: Binding of GST:LRP1-ICD to recombinant human SHP-2 (R&D Systems, 1894-SH-100) was measured using a BIA 3000 optical biosensor (BIAcore AB, Uppsala, Sweden).

Techniques: Incubation, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: The LDL Receptor-Related Protein 1 (LRP1) Regulates the PDGF Signaling Pathway by Binding the Protein Phosphatase SHP-2 and Modulating SHP-2- Mediated PDGF Signaling Events

doi: 10.1371/journal.pone.0070432

Figure Lengend Snippet: (a) WI38 fibroblasts were serum starved overnight and stimulated with PDGF (30 ng/mL) for 2, 5, 10 and 15 minutes at 37°C. Cells were treated with DSP crosslinker for 30 minutes on ice prior to lysis. Lysates were immunoprecipitated with a SHP-2 antibody (sc-280) and western blot analysis was performed using a monoclonal antibody to LRP1, 11H4 (top panel). Loading was controlled by using anti-SHP-2 IgG (bottom panel). As a control, non-immune IgG (IgG) was employed for immunoprecipitation (15 min stimulation with 30 ng/ml PDGF). (b) Immunflourescence studies reveal colocalization of LRP1 and phospho-SHP-2 in fibroblasts stimulated with PDGF-BB. WI38 cells were cultured on glass cover slips, fixed with formaldehyde, and processed for immunofluorescent microscopy as described in “Methods”. The confocal image was taken using 60X oil immersion objective. Box 1 is a representative section from the ‘trailing’ edge of this migrating cell and box 2 is a representative area of the ‘leading’ edge of the cell. (c–e) represent a 3 fold enlarged area from box 2, at the ‘leading’ edge of the cell; (c) green LRP1 staining, (d) red phospho-SHP-2 staining, (e) yellow colocalization of LRP1 and phospho-SHP-2.

Article Snippet: Binding of GST:LRP1-ICD to recombinant human SHP-2 (R&D Systems, 1894-SH-100) was measured using a BIA 3000 optical biosensor (BIAcore AB, Uppsala, Sweden).

Techniques: Lysis, Immunoprecipitation, Western Blot, Cell Culture, Microscopy, Staining

Journal: PLoS ONE

Article Title: The LDL Receptor-Related Protein 1 (LRP1) Regulates the PDGF Signaling Pathway by Binding the Protein Phosphatase SHP-2 and Modulating SHP-2- Mediated PDGF Signaling Events

doi: 10.1371/journal.pone.0070432

Figure Lengend Snippet: Three-dimensional reconstruction from a stack of optical sections of the leading edge of a cell depicted in (box 2). Image is rendered to show sites of colocalization facing forward. Stacks of 5 images 0.2 µm apart were captured with a BioRad confocal (Zeiss) microscope using 100X oil immersion objective; 3D reconstruction was done using Volocity (Improvision) software. LRP1 (green) and phospho-SHP-2 (red).

Article Snippet: Binding of GST:LRP1-ICD to recombinant human SHP-2 (R&D Systems, 1894-SH-100) was measured using a BIA 3000 optical biosensor (BIAcore AB, Uppsala, Sweden).

Techniques: Microscopy, Software

Journal: PLoS ONE

Article Title: The LDL Receptor-Related Protein 1 (LRP1) Regulates the PDGF Signaling Pathway by Binding the Protein Phosphatase SHP-2 and Modulating SHP-2- Mediated PDGF Signaling Events

doi: 10.1371/journal.pone.0070432

Figure Lengend Snippet: LRP expressing (B41) and deficient (LRP −/− ) fibroblasts were seeded onto 5 µm costar transwell filters at 2×10 4 cells per well either in the presence or absence of the SHP-2 inhibitor, NSC-87877. After 3 h incubation at 37°C, either buffer (control) or PDGFB (30 ng/ml) was applied to the bottom chamber and incubation continued for 4 hours at 37°C. After incubation, the topsides of filters were cleared of cells with a cotton swab, and cells remaining on the bottom of the filters were fixed and the nuclei were stained with DAPI overnight. Filters were mounted onto glass slides and nuclei were quantified. (*p = 0.02, **p<0.0001, Students t-test).

Article Snippet: Binding of GST:LRP1-ICD to recombinant human SHP-2 (R&D Systems, 1894-SH-100) was measured using a BIA 3000 optical biosensor (BIAcore AB, Uppsala, Sweden).

Techniques: Expressing, Incubation, Staining