phosphatase inhibitors  (Millipore)


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    Name:
    Leupeptin
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    Catalog Number:
    l8511
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    Structured Review

    Millipore phosphatase inhibitors
    Leupeptin

    https://www.bioz.com/result/phosphatase inhibitors/product/Millipore
    Average 99 stars, based on 2524 article reviews
    Price from $9.99 to $1999.99
    phosphatase inhibitors - by Bioz Stars, 2020-09
    99/100 stars

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    Positive Control:

    Article Title: Mitochondrial NCKX5 regulates melanosomal biogenesis and pigment production
    Article Snippet: .. Cells were further treated with PTU (positive control) and DMSO (negative control) or cultured in the presence of indicated compounds [2 µM CCCP (Sigma-Aldrich), 5 µM oligomycin (Selleck), 0.4 µM rotenone (Sigma-Aldrich), 30 µM 3′,4′-dichlorobenzamil hydrochloride (DBZ; Sigma-Aldrich), 10 μM CGP-37157 (Tocris) or 100 mM leupeptin (Sigma-Aldrich)] for 3 days. ..

    Negative Control:

    Article Title: Mitochondrial NCKX5 regulates melanosomal biogenesis and pigment production
    Article Snippet: .. Cells were further treated with PTU (positive control) and DMSO (negative control) or cultured in the presence of indicated compounds [2 µM CCCP (Sigma-Aldrich), 5 µM oligomycin (Selleck), 0.4 µM rotenone (Sigma-Aldrich), 30 µM 3′,4′-dichlorobenzamil hydrochloride (DBZ; Sigma-Aldrich), 10 μM CGP-37157 (Tocris) or 100 mM leupeptin (Sigma-Aldrich)] for 3 days. ..

    Gas Chromatography:

    Article Title: Mannose 6-Phosphate/Insulin-like Growth Factor-II Receptor Targets the Urokinase Receptor to Lysosomes via a Novel Binding Interaction
    Article Snippet: .. Reagents Glucose 6-phosphate (Glc-6-P), mannose 6-phosphate, and N -propyl-galleat, leupeptin, and pepstatin A were from Sigma Chemical Co. (St. Louis, MO), and Na125 I, d -[2-3 H]mannose, and Pro-mix (l -[35 S]methionine and l -[35 S]cysteine) were from Amersham International (Little Chalfont, UK). .. CNBr-activated Sepharose was purchased from Pharmacia Biotech Sevrage (Uppsala, Sweden), phosphoinositol-specific phospholipase C (PiPLC), Asparaginase-N (Asp-N), and IGF-II were from Boehringer Mannheim GmbH (Mannheim, Germany), and 3,3-dithiobis(sulfosuccinimidylproprionate) (DTSSP) was from Pierce Chemical Co. (Rockford, IL). β-Glucuronidase was purified from the secretions of 13.2.1 cells (a gift of Dr. W. Sly, St. Louis University, St. Louis, MO) as described previously ( ).

    other:

    Article Title: ESCRTs regulate amyloid precursor protein sorting in multivesicular bodies and intracellular amyloid-β accumulation
    Article Snippet: Leupeptin and cycloheximide (Sigma) were used at 50 μg/ml and 40 μg/ml, respectively.

    Cell Culture:

    Article Title: Mitochondrial NCKX5 regulates melanosomal biogenesis and pigment production
    Article Snippet: .. Cells were further treated with PTU (positive control) and DMSO (negative control) or cultured in the presence of indicated compounds [2 µM CCCP (Sigma-Aldrich), 5 µM oligomycin (Selleck), 0.4 µM rotenone (Sigma-Aldrich), 30 µM 3′,4′-dichlorobenzamil hydrochloride (DBZ; Sigma-Aldrich), 10 μM CGP-37157 (Tocris) or 100 mM leupeptin (Sigma-Aldrich)] for 3 days. ..

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  • 99
    Millipore chemicals phosphatase inhibitors salubrinal
    <t>Salubrinal</t> increases PSI-mediated transcriptional activation of ATF4 and CHOP, promotes XBP1 mRNA splicing and reduces cell viability. (A) K562 cells stably expressing a 5′ATF4.GFP or CHOP::GFP reporter gene, were challenged for 15 h with 5 nM PSI, 10 µM salubrinal and 2 mM VPA as indicated. During the last 30 min of the incubation 100 nM mitotracker orange CMXRos was added to determine mitochondrial transmembrane potential ΔΨ. Fluorescence changes were monitored by FACS analysis. The pan-caspase inhibitor Q-VD-OPH was used at a concentration of 5 µM. Scatter plots are representatives from three independent experiments each performed in triplicate with similar results and show the distribution of cells with increased GFP fluorescence, indicative for transcriptional activation of ATF4 and CHOP relative to the percentage of cells with decreased ΔΨ, indicative for reduced viability and the onset of apoptosis. (B) Numerical evaluation of results shown in (A); values represent % cells with increased GFP- and decreased CMXRos–related fluorescence, respectively. Data were obtained from one out of three independent experiment each performed in triplicate; error bars represent deviation from the mean. (C) XBP1 mRNA splicing by salubrinal and PSI. RT-PCR analysis with XBP1 or ß-actin specific primers of total RNA extracted from K562 cells, that were incubated with 5 nM PSI, 2 mM VPA and 10 µM salubrinal for 15 h as indicated. XBP H : hybrid XBP1; XBP1 U : unspliced XBP1; XBP1 S : spliced XBP1.
    Chemicals Phosphatase Inhibitors Salubrinal, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 1 article reviews
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    85
    Millipore serine threonine phosphatase inhibitors okadaic acid
    Effect of phosphatase inhibitors on PP1, PP2A, and PP2B enzymatic activity. Sperm were selected and re-suspended in NCM. Each phosphatase activity was measured in sperm extracts as indicated in Materials and Methods in the absence of inhibitors (black bars) and then in the presence of 10 nM <t>okadaic</t> acid (white bar); 40 µg/ml I2 (grey bar); 90 nM endothall (dotted bar); 0.1 nM okadaic acid (light grey bar); or 0.1 nM deltamethrin (striped bar). Results were obtained from five different donors and are expressed as the mean±SEM of forty measurements every 2 min. * p
    Serine Threonine Phosphatase Inhibitors Okadaic Acid, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore phosphatase inhibitors okadaic acid oa
    AVP washout in the presence of phosphatase inhibitors preserves the AQP2 and 14-3-3 interaction. mpkCCD 14 cells were acutely stimulated with dDAVP followed by a washout period in the presence or absence of the inhibitors <t>okadaic</t> acid and calyculin A.
    Phosphatase Inhibitors Okadaic Acid Oa, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phosphatase inhibitors okadaic acid oa/product/Millipore
    Average 90 stars, based on 1 article reviews
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    88
    Millipore shp 2 inhibitor
    <t>SHP-2</t> and LRP1 co-immunoprecipitate and co-localize in fibroblasts following PDGF stimulation. (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.
    Shp 2 Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Salubrinal increases PSI-mediated transcriptional activation of ATF4 and CHOP, promotes XBP1 mRNA splicing and reduces cell viability. (A) K562 cells stably expressing a 5′ATF4.GFP or CHOP::GFP reporter gene, were challenged for 15 h with 5 nM PSI, 10 µM salubrinal and 2 mM VPA as indicated. During the last 30 min of the incubation 100 nM mitotracker orange CMXRos was added to determine mitochondrial transmembrane potential ΔΨ. Fluorescence changes were monitored by FACS analysis. The pan-caspase inhibitor Q-VD-OPH was used at a concentration of 5 µM. Scatter plots are representatives from three independent experiments each performed in triplicate with similar results and show the distribution of cells with increased GFP fluorescence, indicative for transcriptional activation of ATF4 and CHOP relative to the percentage of cells with decreased ΔΨ, indicative for reduced viability and the onset of apoptosis. (B) Numerical evaluation of results shown in (A); values represent % cells with increased GFP- and decreased CMXRos–related fluorescence, respectively. Data were obtained from one out of three independent experiment each performed in triplicate; error bars represent deviation from the mean. (C) XBP1 mRNA splicing by salubrinal and PSI. RT-PCR analysis with XBP1 or ß-actin specific primers of total RNA extracted from K562 cells, that were incubated with 5 nM PSI, 2 mM VPA and 10 µM salubrinal for 15 h as indicated. XBP H : hybrid XBP1; XBP1 U : unspliced XBP1; XBP1 S : spliced XBP1.

    Journal: PLoS ONE

    Article Title: Synergistic Apoptosis Induction in Leukemic Cells by the Phosphatase Inhibitor Salubrinal and Proteasome Inhibitors

    doi: 10.1371/journal.pone.0004161

    Figure Lengend Snippet: Salubrinal increases PSI-mediated transcriptional activation of ATF4 and CHOP, promotes XBP1 mRNA splicing and reduces cell viability. (A) K562 cells stably expressing a 5′ATF4.GFP or CHOP::GFP reporter gene, were challenged for 15 h with 5 nM PSI, 10 µM salubrinal and 2 mM VPA as indicated. During the last 30 min of the incubation 100 nM mitotracker orange CMXRos was added to determine mitochondrial transmembrane potential ΔΨ. Fluorescence changes were monitored by FACS analysis. The pan-caspase inhibitor Q-VD-OPH was used at a concentration of 5 µM. Scatter plots are representatives from three independent experiments each performed in triplicate with similar results and show the distribution of cells with increased GFP fluorescence, indicative for transcriptional activation of ATF4 and CHOP relative to the percentage of cells with decreased ΔΨ, indicative for reduced viability and the onset of apoptosis. (B) Numerical evaluation of results shown in (A); values represent % cells with increased GFP- and decreased CMXRos–related fluorescence, respectively. Data were obtained from one out of three independent experiment each performed in triplicate; error bars represent deviation from the mean. (C) XBP1 mRNA splicing by salubrinal and PSI. RT-PCR analysis with XBP1 or ß-actin specific primers of total RNA extracted from K562 cells, that were incubated with 5 nM PSI, 2 mM VPA and 10 µM salubrinal for 15 h as indicated. XBP H : hybrid XBP1; XBP1 U : unspliced XBP1; XBP1 S : spliced XBP1.

    Article Snippet: Chemicals Phosphatase inhibitors salubrinal and cantharidine were purchased from Calbiochem; sodium valproate was obtained from Sigma (Deisenhofen, Germany).

    Techniques: Activation Assay, Stable Transfection, Expressing, Incubation, Fluorescence, FACS, Concentration Assay, Reverse Transcription Polymerase Chain Reaction

    The amplification of PSI-mediated apoptosis by salubrinal is associated with activation of caspase-3 and 8 and simultaneous upregulation of Bim and Mcl-1. (A) Combined caspase 3 and 7 activities in K562 cells treated with 5 nM PSI, 10 µM salubrinal and 2 mM VPA as indicated for 18 h. (B) Caspase-8 activities in K562 cell lysates treated as in (A). Results were expressed as relative fluorescence units of the fluorescence determined at 360 nm ex /460 nm em (RFU; mean±SD). (C) Western blot analysis. Whole-cell lysates were prepared from K562 cells treated with 5 nM PSI, 10 µM salubrinal, 2 mM VPA and 5 µM Q-VD-OPH as pan-caspase inhibitor as indicated, separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were subsequently probed for cleaved caspase-3, 8 and 9 and for PARP. In (D) cell lysates were analyzed in an analogous fashion by sequential probing with antibodies reacting against epitopes specfic for Bim, Mcl-1, Bax, Bak, Bad and Bcl-xL. ß-actin and ß-tubulin were used as loading controls.

    Journal: PLoS ONE

    Article Title: Synergistic Apoptosis Induction in Leukemic Cells by the Phosphatase Inhibitor Salubrinal and Proteasome Inhibitors

    doi: 10.1371/journal.pone.0004161

    Figure Lengend Snippet: The amplification of PSI-mediated apoptosis by salubrinal is associated with activation of caspase-3 and 8 and simultaneous upregulation of Bim and Mcl-1. (A) Combined caspase 3 and 7 activities in K562 cells treated with 5 nM PSI, 10 µM salubrinal and 2 mM VPA as indicated for 18 h. (B) Caspase-8 activities in K562 cell lysates treated as in (A). Results were expressed as relative fluorescence units of the fluorescence determined at 360 nm ex /460 nm em (RFU; mean±SD). (C) Western blot analysis. Whole-cell lysates were prepared from K562 cells treated with 5 nM PSI, 10 µM salubrinal, 2 mM VPA and 5 µM Q-VD-OPH as pan-caspase inhibitor as indicated, separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were subsequently probed for cleaved caspase-3, 8 and 9 and for PARP. In (D) cell lysates were analyzed in an analogous fashion by sequential probing with antibodies reacting against epitopes specfic for Bim, Mcl-1, Bax, Bak, Bad and Bcl-xL. ß-actin and ß-tubulin were used as loading controls.

    Article Snippet: Chemicals Phosphatase inhibitors salubrinal and cantharidine were purchased from Calbiochem; sodium valproate was obtained from Sigma (Deisenhofen, Germany).

    Techniques: Amplification, Activation Assay, Fluorescence, Western Blot, SDS Page

    Salubrinal synergistically interacts with the proteasome inhibitor PSI to induce cell death in K562 chronic myeloid leukemia cells. (A) K562 cells were exposed to 5 nM PSI for 18 h either alone or in combination with 2 mM valproic acid and 5 or 10 µM salubrinal as indicated. The protein synthesis inhibitor cycloheximide (CHX) was used at 1 µg/ml. Apoptosis induction was assessed by propidium iodide staining and fluorescence-activated cell sorting of cells with a subdiploid (G

    Journal: PLoS ONE

    Article Title: Synergistic Apoptosis Induction in Leukemic Cells by the Phosphatase Inhibitor Salubrinal and Proteasome Inhibitors

    doi: 10.1371/journal.pone.0004161

    Figure Lengend Snippet: Salubrinal synergistically interacts with the proteasome inhibitor PSI to induce cell death in K562 chronic myeloid leukemia cells. (A) K562 cells were exposed to 5 nM PSI for 18 h either alone or in combination with 2 mM valproic acid and 5 or 10 µM salubrinal as indicated. The protein synthesis inhibitor cycloheximide (CHX) was used at 1 µg/ml. Apoptosis induction was assessed by propidium iodide staining and fluorescence-activated cell sorting of cells with a subdiploid (G

    Article Snippet: Chemicals Phosphatase inhibitors salubrinal and cantharidine were purchased from Calbiochem; sodium valproate was obtained from Sigma (Deisenhofen, Germany).

    Techniques: Staining, Fluorescence, FACS

    The salubrinal-enhanced toxicity is not restricted to PSI and is recapitulated by the PP1/PP2A inhibitor cantharidin. (A) K562 cells were exposed to 100 nM of the proteasome inhibitor MG132, 10 µM salubrinal and VPA for 18 h as indicated, after which apoptosis induction was assessed by fluorescence activated cell sorting of cells with a subdiploid (G

    Journal: PLoS ONE

    Article Title: Synergistic Apoptosis Induction in Leukemic Cells by the Phosphatase Inhibitor Salubrinal and Proteasome Inhibitors

    doi: 10.1371/journal.pone.0004161

    Figure Lengend Snippet: The salubrinal-enhanced toxicity is not restricted to PSI and is recapitulated by the PP1/PP2A inhibitor cantharidin. (A) K562 cells were exposed to 100 nM of the proteasome inhibitor MG132, 10 µM salubrinal and VPA for 18 h as indicated, after which apoptosis induction was assessed by fluorescence activated cell sorting of cells with a subdiploid (G

    Article Snippet: Chemicals Phosphatase inhibitors salubrinal and cantharidine were purchased from Calbiochem; sodium valproate was obtained from Sigma (Deisenhofen, Germany).

    Techniques: Fluorescence, FACS

    PP1γ and PP2A phosphatase activities are not affected by salubrinal. Phosphatase activities were determined on immunoprecipitates of the corresponding phosphatases as described in Materials and Methods . Following treatment phosphatases were immunoprecipitated and the activities of the corresponding phosphatases measured Phosphatase activities are given as percent change relative to the control (DMSO treated cells). Results shown are the mean±SEM of 4 independent experiments for each phosphatase.

    Journal: PLoS ONE

    Article Title: Synergistic Apoptosis Induction in Leukemic Cells by the Phosphatase Inhibitor Salubrinal and Proteasome Inhibitors

    doi: 10.1371/journal.pone.0004161

    Figure Lengend Snippet: PP1γ and PP2A phosphatase activities are not affected by salubrinal. Phosphatase activities were determined on immunoprecipitates of the corresponding phosphatases as described in Materials and Methods . Following treatment phosphatases were immunoprecipitated and the activities of the corresponding phosphatases measured Phosphatase activities are given as percent change relative to the control (DMSO treated cells). Results shown are the mean±SEM of 4 independent experiments for each phosphatase.

    Article Snippet: Chemicals Phosphatase inhibitors salubrinal and cantharidine were purchased from Calbiochem; sodium valproate was obtained from Sigma (Deisenhofen, Germany).

    Techniques: Immunoprecipitation

    Stimulation of PSI-mediated apoptosis is delayed by overexpression of Bcl-xL and Flag-crmA. (A) Whole-cell lysates prepared from K562 cells (ATCC) stably expressing either Flag-crmA or Bcl-xL was subjected to Western blot analysis using antibodies reacting against Bcl-xL or the Flag epitope. ß-tubulin: loading control. (B) Control, Flag-crmA and Bcl-xL overexpressing K562 cells were exposed for 24 h to 20 nM PSI and 20 µM salubrinal as indicated after which apoptosis induction was determined as described in Materials and Methods (mean±SD of 3 determinations).

    Journal: PLoS ONE

    Article Title: Synergistic Apoptosis Induction in Leukemic Cells by the Phosphatase Inhibitor Salubrinal and Proteasome Inhibitors

    doi: 10.1371/journal.pone.0004161

    Figure Lengend Snippet: Stimulation of PSI-mediated apoptosis is delayed by overexpression of Bcl-xL and Flag-crmA. (A) Whole-cell lysates prepared from K562 cells (ATCC) stably expressing either Flag-crmA or Bcl-xL was subjected to Western blot analysis using antibodies reacting against Bcl-xL or the Flag epitope. ß-tubulin: loading control. (B) Control, Flag-crmA and Bcl-xL overexpressing K562 cells were exposed for 24 h to 20 nM PSI and 20 µM salubrinal as indicated after which apoptosis induction was determined as described in Materials and Methods (mean±SD of 3 determinations).

    Article Snippet: Chemicals Phosphatase inhibitors salubrinal and cantharidine were purchased from Calbiochem; sodium valproate was obtained from Sigma (Deisenhofen, Germany).

    Techniques: Over Expression, Stable Transfection, Expressing, Western Blot, FLAG-tag

    Stimulation of PSI-mediated apoptosis is not inhibited by overexpression of the transdominant eIF2α S51A mutant (A) Whole-cell lysates from K562 cells (ATCC) stably expressing myc-tagged eIF2α S51A (clone 3, 8) or a non-expressing control clone (clone 11) were subjected to Western blot analysis using antibodies reacting against the myc epitope or full-length eIF2α. ß-tubulin served as loading control. Myc-tagged eIF2α S51A is marked by an asterisk, endogenous eIF2α by an arrowhead. (B) Control and eIF2α-overexpressing K562 cells were exposed to PSI and salubrinal as indicated and the extent of apoptosis induction determined as described in Materials and Methods (mean±SD of 3 measurements).

    Journal: PLoS ONE

    Article Title: Synergistic Apoptosis Induction in Leukemic Cells by the Phosphatase Inhibitor Salubrinal and Proteasome Inhibitors

    doi: 10.1371/journal.pone.0004161

    Figure Lengend Snippet: Stimulation of PSI-mediated apoptosis is not inhibited by overexpression of the transdominant eIF2α S51A mutant (A) Whole-cell lysates from K562 cells (ATCC) stably expressing myc-tagged eIF2α S51A (clone 3, 8) or a non-expressing control clone (clone 11) were subjected to Western blot analysis using antibodies reacting against the myc epitope or full-length eIF2α. ß-tubulin served as loading control. Myc-tagged eIF2α S51A is marked by an asterisk, endogenous eIF2α by an arrowhead. (B) Control and eIF2α-overexpressing K562 cells were exposed to PSI and salubrinal as indicated and the extent of apoptosis induction determined as described in Materials and Methods (mean±SD of 3 measurements).

    Article Snippet: Chemicals Phosphatase inhibitors salubrinal and cantharidine were purchased from Calbiochem; sodium valproate was obtained from Sigma (Deisenhofen, Germany).

    Techniques: Over Expression, Mutagenesis, Stable Transfection, Expressing, Western Blot

    Exposure to cycloheximide (CHX) results in global inhibition of protein synthesis and abrogation of stress kinase signaling. (A) Translational inhibition by CHX in K562 cells was monitored by labeling nascent proteins with the methionine analog L-azidohomoalanine (AHA). Growth in the presence of AHA resulted in extensive labeling of proteins (lane 2), which was completely abrogated by CHX (lane 3). PSI (5 nM) slightly reduced protein synthesis (lane 4). Proteins from cells grown in the presence of methionine served as control for the labeling reaction (lane 1). Lower panel: ß-tubulin as loading control. (B, C) Whole cell lysates from K562 exposed to 5 nM PSI, 10 µM salubrinal, 2 mM VPA and 1 µg/ml CHX were subjected to SDS-PAGE and Western blot analysis as indicated. ß-tubulin or ß-actin antibodies were used to demonstrate equal loading.

    Journal: PLoS ONE

    Article Title: Synergistic Apoptosis Induction in Leukemic Cells by the Phosphatase Inhibitor Salubrinal and Proteasome Inhibitors

    doi: 10.1371/journal.pone.0004161

    Figure Lengend Snippet: Exposure to cycloheximide (CHX) results in global inhibition of protein synthesis and abrogation of stress kinase signaling. (A) Translational inhibition by CHX in K562 cells was monitored by labeling nascent proteins with the methionine analog L-azidohomoalanine (AHA). Growth in the presence of AHA resulted in extensive labeling of proteins (lane 2), which was completely abrogated by CHX (lane 3). PSI (5 nM) slightly reduced protein synthesis (lane 4). Proteins from cells grown in the presence of methionine served as control for the labeling reaction (lane 1). Lower panel: ß-tubulin as loading control. (B, C) Whole cell lysates from K562 exposed to 5 nM PSI, 10 µM salubrinal, 2 mM VPA and 1 µg/ml CHX were subjected to SDS-PAGE and Western blot analysis as indicated. ß-tubulin or ß-actin antibodies were used to demonstrate equal loading.

    Article Snippet: Chemicals Phosphatase inhibitors salubrinal and cantharidine were purchased from Calbiochem; sodium valproate was obtained from Sigma (Deisenhofen, Germany).

    Techniques: Inhibition, Labeling, SDS Page, Western Blot

    Salubrinal is nontoxic, but elicits a G2/M arrest in K562 cells. (A) K562 cells in a 96 well plate were exposed to variable concentrations of salubrinal for 15 h after which viability of cells was assessed by incubation with WST-1 reagent. Soluble formazan formation was determined by absorption measurement at 450 nm. Cells incubated with DMSO served as solvent control; incubation with 5 nM PSI and 2 mM VPA was a control for effective apoptosis induction. Data are presented as means±SD (n = 3); (B) Whole-cell lysates were prepared from cells that were treated with increasing concentrations of salubrinal, separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were sequentially probed for P-eIF2α, eIF2α, CHOP, PARP, KDEL, GADD34 and ß-tubulin. Incubation with 2 µM thapsigargin served as positive control for the induction of ER-stress. The extent of PARP cleavage was determined by densitometry and is provided as fold increase in brakets below the Western blot for PARP. Shown are representative blots obtained from at least two independent experiments with similar results (C) K562 cells exposed to 5 nM PSI, 10 µM salubrinal and 2 mM VPA as indicated were stained with propidium iodide and analyzed by fluorescence-activated cell sorting. Histograms are representative examples. Cell cycle distribution values were derived by gating for viable cells followed by application of Modfit 3.0 software (Becton Dickinson). Data are presented as means±SD (n = 3).

    Journal: PLoS ONE

    Article Title: Synergistic Apoptosis Induction in Leukemic Cells by the Phosphatase Inhibitor Salubrinal and Proteasome Inhibitors

    doi: 10.1371/journal.pone.0004161

    Figure Lengend Snippet: Salubrinal is nontoxic, but elicits a G2/M arrest in K562 cells. (A) K562 cells in a 96 well plate were exposed to variable concentrations of salubrinal for 15 h after which viability of cells was assessed by incubation with WST-1 reagent. Soluble formazan formation was determined by absorption measurement at 450 nm. Cells incubated with DMSO served as solvent control; incubation with 5 nM PSI and 2 mM VPA was a control for effective apoptosis induction. Data are presented as means±SD (n = 3); (B) Whole-cell lysates were prepared from cells that were treated with increasing concentrations of salubrinal, separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were sequentially probed for P-eIF2α, eIF2α, CHOP, PARP, KDEL, GADD34 and ß-tubulin. Incubation with 2 µM thapsigargin served as positive control for the induction of ER-stress. The extent of PARP cleavage was determined by densitometry and is provided as fold increase in brakets below the Western blot for PARP. Shown are representative blots obtained from at least two independent experiments with similar results (C) K562 cells exposed to 5 nM PSI, 10 µM salubrinal and 2 mM VPA as indicated were stained with propidium iodide and analyzed by fluorescence-activated cell sorting. Histograms are representative examples. Cell cycle distribution values were derived by gating for viable cells followed by application of Modfit 3.0 software (Becton Dickinson). Data are presented as means±SD (n = 3).

    Article Snippet: Chemicals Phosphatase inhibitors salubrinal and cantharidine were purchased from Calbiochem; sodium valproate was obtained from Sigma (Deisenhofen, Germany).

    Techniques: Incubation, SDS Page, Positive Control, Western Blot, Staining, Fluorescence, FACS, Derivative Assay, Software

    Salubrinal promotes the cytotoxic effects elicited by the ER stressor thapsigargin. K562 cells (10 5 /ml) were exposed for 18 h to thapsigargin (0.5–2.0 µM) either alone or in combination with 10 µM salubrinal. Apoptosis was determined by fluorescence activated cell sorting of cells with a subdiploid (G

    Journal: PLoS ONE

    Article Title: Synergistic Apoptosis Induction in Leukemic Cells by the Phosphatase Inhibitor Salubrinal and Proteasome Inhibitors

    doi: 10.1371/journal.pone.0004161

    Figure Lengend Snippet: Salubrinal promotes the cytotoxic effects elicited by the ER stressor thapsigargin. K562 cells (10 5 /ml) were exposed for 18 h to thapsigargin (0.5–2.0 µM) either alone or in combination with 10 µM salubrinal. Apoptosis was determined by fluorescence activated cell sorting of cells with a subdiploid (G

    Article Snippet: Chemicals Phosphatase inhibitors salubrinal and cantharidine were purchased from Calbiochem; sodium valproate was obtained from Sigma (Deisenhofen, Germany).

    Techniques: Fluorescence, FACS

    Acceleration of cell death induction by salubrinal is inhibited by the pan-caspase-inhibitor Q-VD-OPH. (A, B) K562 cells were exposed to 5 nM PSI in conjunction with 10 µM salubrinal and/or 2 mM VPA for the indicated intervals after which cells were monitored for apoptosis by FACS analysis. The pan-caspase inhibitor Q-VD-OPH was added simultaneously with the other compounds (5 µM final concentration). Data represent the means±SD of an assay performed in triplicate out of two independent experiments with similar results. DMSO control (open circles), PSI 5 nM (open squares), salubrinal 10 µM open triangles), PSI+salubrinal (filled triangles), PSI+salubrinal+Q-VD-OPH (filled inverted triangles), PSI+VPA 2 mM (filled squares), PSI+VPA+salubrinal (diamonds), PSI+VPA+salubrinal+QVD-OPH (filled circles); (C) Whole-cell lysates were prepared from cells incubated with 5 nM PSI, 2 mM VPA and 10 µM salubrinal as indicated, separated by SDS-Page and transferred to nitrocellulose membranes. The membranes were sequentially probed for PARP, polyubiquitylated proteins and ß-tubulin.

    Journal: PLoS ONE

    Article Title: Synergistic Apoptosis Induction in Leukemic Cells by the Phosphatase Inhibitor Salubrinal and Proteasome Inhibitors

    doi: 10.1371/journal.pone.0004161

    Figure Lengend Snippet: Acceleration of cell death induction by salubrinal is inhibited by the pan-caspase-inhibitor Q-VD-OPH. (A, B) K562 cells were exposed to 5 nM PSI in conjunction with 10 µM salubrinal and/or 2 mM VPA for the indicated intervals after which cells were monitored for apoptosis by FACS analysis. The pan-caspase inhibitor Q-VD-OPH was added simultaneously with the other compounds (5 µM final concentration). Data represent the means±SD of an assay performed in triplicate out of two independent experiments with similar results. DMSO control (open circles), PSI 5 nM (open squares), salubrinal 10 µM open triangles), PSI+salubrinal (filled triangles), PSI+salubrinal+Q-VD-OPH (filled inverted triangles), PSI+VPA 2 mM (filled squares), PSI+VPA+salubrinal (diamonds), PSI+VPA+salubrinal+QVD-OPH (filled circles); (C) Whole-cell lysates were prepared from cells incubated with 5 nM PSI, 2 mM VPA and 10 µM salubrinal as indicated, separated by SDS-Page and transferred to nitrocellulose membranes. The membranes were sequentially probed for PARP, polyubiquitylated proteins and ß-tubulin.

    Article Snippet: Chemicals Phosphatase inhibitors salubrinal and cantharidine were purchased from Calbiochem; sodium valproate was obtained from Sigma (Deisenhofen, Germany).

    Techniques: FACS, Concentration Assay, Incubation, SDS Page

    Effect of phosphatase inhibitors on PP1, PP2A, and PP2B enzymatic activity. Sperm were selected and re-suspended in NCM. Each phosphatase activity was measured in sperm extracts as indicated in Materials and Methods in the absence of inhibitors (black bars) and then in the presence of 10 nM okadaic acid (white bar); 40 µg/ml I2 (grey bar); 90 nM endothall (dotted bar); 0.1 nM okadaic acid (light grey bar); or 0.1 nM deltamethrin (striped bar). Results were obtained from five different donors and are expressed as the mean±SEM of forty measurements every 2 min. * p

    Journal: PLoS ONE

    Article Title: Protein Phosphatases Decrease Their Activity during Capacitation: A New Requirement for This Event

    doi: 10.1371/journal.pone.0081286

    Figure Lengend Snippet: Effect of phosphatase inhibitors on PP1, PP2A, and PP2B enzymatic activity. Sperm were selected and re-suspended in NCM. Each phosphatase activity was measured in sperm extracts as indicated in Materials and Methods in the absence of inhibitors (black bars) and then in the presence of 10 nM okadaic acid (white bar); 40 µg/ml I2 (grey bar); 90 nM endothall (dotted bar); 0.1 nM okadaic acid (light grey bar); or 0.1 nM deltamethrin (striped bar). Results were obtained from five different donors and are expressed as the mean±SEM of forty measurements every 2 min. * p

    Article Snippet: The serine-threonine phosphatase inhibitors okadaic acid, deltamethrin, and calmodulin were obtained from Calbiochem Corporation (La Jolla, CA).

    Techniques: Activity Assay

    Effect of protein phosphatase inhibitors on human sperm capacitation. Human sperm were incubated in NCM (A) or RCM (B) for 120 min, and the capacitation pattern was evaluated using the CTC assay, as described in the Materials and Methods . The inhibitors used were okadaic acid (black squares), deltamethrin (green diamonds), and endothall (blue triangles). Results were obtained from five different donors and were expressed as the mean±SEM of the percentage of B pattern. The arrow indicates the addition of BSA and bicarbonate. * p

    Journal: PLoS ONE

    Article Title: Protein Phosphatases Decrease Their Activity during Capacitation: A New Requirement for This Event

    doi: 10.1371/journal.pone.0081286

    Figure Lengend Snippet: Effect of protein phosphatase inhibitors on human sperm capacitation. Human sperm were incubated in NCM (A) or RCM (B) for 120 min, and the capacitation pattern was evaluated using the CTC assay, as described in the Materials and Methods . The inhibitors used were okadaic acid (black squares), deltamethrin (green diamonds), and endothall (blue triangles). Results were obtained from five different donors and were expressed as the mean±SEM of the percentage of B pattern. The arrow indicates the addition of BSA and bicarbonate. * p

    Article Snippet: The serine-threonine phosphatase inhibitors okadaic acid, deltamethrin, and calmodulin were obtained from Calbiochem Corporation (La Jolla, CA).

    Techniques: Incubation

    Effect of phosphatase inhibitors on phosphorylation of threonine residues of human sperm proteins. Sperm were selected in NCM and then re-suspended in RCM in the absence (RCM) or in the presence of 10 nM okadaic acid (OA), 90 nM endothall (E), or 0.1 nM deltamethrin (D), for 1 min. Then, the samples were processed for western blot analysis with a monoclonal anti-p-Thr antibody. Molecular mass of selected bands is indicated at the right. The lower panels indicate load control with anti β-tubulin antibody. The immunoblots shown are representative of three different experiments with three different donors.

    Journal: PLoS ONE

    Article Title: Protein Phosphatases Decrease Their Activity during Capacitation: A New Requirement for This Event

    doi: 10.1371/journal.pone.0081286

    Figure Lengend Snippet: Effect of phosphatase inhibitors on phosphorylation of threonine residues of human sperm proteins. Sperm were selected in NCM and then re-suspended in RCM in the absence (RCM) or in the presence of 10 nM okadaic acid (OA), 90 nM endothall (E), or 0.1 nM deltamethrin (D), for 1 min. Then, the samples were processed for western blot analysis with a monoclonal anti-p-Thr antibody. Molecular mass of selected bands is indicated at the right. The lower panels indicate load control with anti β-tubulin antibody. The immunoblots shown are representative of three different experiments with three different donors.

    Article Snippet: The serine-threonine phosphatase inhibitors okadaic acid, deltamethrin, and calmodulin were obtained from Calbiochem Corporation (La Jolla, CA).

    Techniques: Western Blot

    AVP washout in the presence of phosphatase inhibitors preserves the AQP2 and 14-3-3 interaction. mpkCCD 14 cells were acutely stimulated with dDAVP followed by a washout period in the presence or absence of the inhibitors okadaic acid and calyculin A.

    Journal: The Journal of Biological Chemistry

    Article Title: Regulation of the Water Channel Aquaporin-2 via 14-3-3θ and -ζ *

    doi: 10.1074/jbc.M115.691121

    Figure Lengend Snippet: AVP washout in the presence of phosphatase inhibitors preserves the AQP2 and 14-3-3 interaction. mpkCCD 14 cells were acutely stimulated with dDAVP followed by a washout period in the presence or absence of the inhibitors okadaic acid and calyculin A.

    Article Snippet: The phosphatase inhibitors okadaic acid (OA) and calyculin A (CA) were from Calbiochem.

    Techniques:

    SHP-2 and LRP1 co-immunoprecipitate and co-localize in fibroblasts following PDGF stimulation. (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.

    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: SHP-2 and LRP1 co-immunoprecipitate and co-localize in fibroblasts following PDGF stimulation. (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: Cells were pretreated for 3 hours with 30 µM SHP-2 inhibitor, NSC-87877 (Millipore, 565851).

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

    Co-localization of LRP1 and phospho-LRP1 in WI-38 fibroblasts. Three-dimensional reconstruction from a stack of optical sections of the leading edge of a cell depicted in Fig. 6 (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).

    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: Co-localization of LRP1 and phospho-LRP1 in WI-38 fibroblasts. Three-dimensional reconstruction from a stack of optical sections of the leading edge of a cell depicted in Fig. 6 (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: Cells were pretreated for 3 hours with 30 µM SHP-2 inhibitor, NSC-87877 (Millipore, 565851).

    Techniques: Microscopy, Software

    LRP1 modulates SHP-2-mediated migration in response to PDGF-B. 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

    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: LRP1 modulates SHP-2-mediated migration in response to PDGF-B. 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

    Article Snippet: Cells were pretreated for 3 hours with 30 µM SHP-2 inhibitor, NSC-87877 (Millipore, 565851).

    Techniques: Migration, Expressing, Incubation, Staining

    Phosphorylated LRP1-ICD interacts with SHP-2 with high affinity. (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

    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: Phosphorylated LRP1-ICD interacts with SHP-2 with high affinity. (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

    Article Snippet: Cells were pretreated for 3 hours with 30 µM SHP-2 inhibitor, NSC-87877 (Millipore, 565851).

    Techniques: Incubation, SDS Page, Staining

    pPDGFRβ kinase domain and pLRP1-ICD compete for SHP-2 binding. (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

    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: pPDGFRβ kinase domain and pLRP1-ICD compete for SHP-2 binding. (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

    Article Snippet: Cells were pretreated for 3 hours with 30 µM SHP-2 inhibitor, NSC-87877 (Millipore, 565851).

    Techniques: Binding Assay, Incubation, Concentration Assay

    Quantitative analysis of interaction of the PDGFRβ kinase domain with SHP-2. 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).

    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: Quantitative analysis of interaction of the PDGFRβ kinase domain with SHP-2. 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: Cells were pretreated for 3 hours with 30 µM SHP-2 inhibitor, NSC-87877 (Millipore, 565851).

    Techniques: Incubation, Binding Assay

    PDGF-mediated tyrosine phosphorylation of SHP-2 occurs within endosomes. (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.

    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: PDGF-mediated tyrosine phosphorylation of SHP-2 occurs within endosomes. (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: Cells were pretreated for 3 hours with 30 µM SHP-2 inhibitor, NSC-87877 (Millipore, 565851).

    Techniques: Incubation, SDS Page

    PDGFRβ kinase domain directly mediates the phosphorylation of SHP-2. (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

    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: PDGFRβ kinase domain directly mediates the phosphorylation of SHP-2. (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

    Article Snippet: Cells were pretreated for 3 hours with 30 µM SHP-2 inhibitor, NSC-87877 (Millipore, 565851).

    Techniques: Purification, Incubation, Recombinant, SDS Page, Activity Assay, Phosphatase Assay