pp2  (Millipore)


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    Structured Review

    Millipore pp2
    PP2

    https://www.bioz.com/result/pp2/product/Millipore
    Average 95 stars, based on 65 article reviews
    Price from $9.99 to $1999.99
    pp2 - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Cyclic stretch disrupts apical junctional complexes in Caco-2 cell monolayers by a JNK-2-, c-Src-, and MLCK-dependent mechanism"

    Article Title: Cyclic stretch disrupts apical junctional complexes in Caco-2 cell monolayers by a JNK-2-, c-Src-, and MLCK-dependent mechanism

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    doi: 10.1152/ajpgi.00396.2013

    c-Src is involved in the mechanism of stretch-induced tight junction and adherens junction disruption. Caco-2 cell monolayers on Flexcell plates were pretreated with PP2 for 20 min prior to application of cyclic stretch for varying times. A and B : 2 h
    Figure Legend Snippet: c-Src is involved in the mechanism of stretch-induced tight junction and adherens junction disruption. Caco-2 cell monolayers on Flexcell plates were pretreated with PP2 for 20 min prior to application of cyclic stretch for varying times. A and B : 2 h

    Techniques Used:

    Inhibition of JNK and Src kinase attenuates stretch-induced reorganization of actin cytoskeleton. Caco-2 cell monolayers on Flexcell plates were pretreated with SP600125 (SP) or PP2 prior to application of cyclic stretch for 2 h. Fixed cell monolayers
    Figure Legend Snippet: Inhibition of JNK and Src kinase attenuates stretch-induced reorganization of actin cytoskeleton. Caco-2 cell monolayers on Flexcell plates were pretreated with SP600125 (SP) or PP2 prior to application of cyclic stretch for 2 h. Fixed cell monolayers

    Techniques Used: Inhibition

    2) Product Images from "An RPTPα/Src family kinase/Rap1 signaling module recruits myosin IIB to support contractile tension at apical E-cadherin junctions"

    Article Title: An RPTPα/Src family kinase/Rap1 signaling module recruits myosin IIB to support contractile tension at apical E-cadherin junctions

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E14-07-1223

    Rap1 is a downstream target of junctional SFK signaling. (A) Subcellular distribution of GFP-Rap1WT in Control and PP2-treated cells analyzed by live-cell imaging. (B) FRET/YFP emission ratio images of cells transfected with Raichu-Rap1 FRET biosensor in control cells, PP2-treated cells, and cells transfected with RPTPα siRNA. (C) Quantitative analysis of Rap1 activity (FRET/YFP) emission ratios at the ZA of control, PP2-treated, RPTPα KD, and PP2-treated RPTPα KD cells. Data in C are means ± SEM for at least 25 images (∼90–100 cells) per condition. **, p
    Figure Legend Snippet: Rap1 is a downstream target of junctional SFK signaling. (A) Subcellular distribution of GFP-Rap1WT in Control and PP2-treated cells analyzed by live-cell imaging. (B) FRET/YFP emission ratio images of cells transfected with Raichu-Rap1 FRET biosensor in control cells, PP2-treated cells, and cells transfected with RPTPα siRNA. (C) Quantitative analysis of Rap1 activity (FRET/YFP) emission ratios at the ZA of control, PP2-treated, RPTPα KD, and PP2-treated RPTPα KD cells. Data in C are means ± SEM for at least 25 images (∼90–100 cells) per condition. **, p

    Techniques Used: Live Cell Imaging, Transfection, Activity Assay

    c-Src and c-Yes contribute to junctional SFK signaling in MCF-7 cells. (A and B) Quantitative immunofluorescence analysis of NMIIB at the ZA in MCF-7 cells treated with control, PP2, RPTPα siRNA, and RPTPα siRNA+PP2 (25 μM, 1 h). Representative images of E-cadherin and NMIIB in control and PP2-treated cells (A) and quantitative line-scan analysis of NMIIB fluorescence at junctions (B). (C) Normalized initial recoil values for laser nanoscissor experiments performed in MCF-7 cells treated with control, PP2 (25 μM, 1 h) and Y-27632 (30 μM, 1 h). (D) Immunofluorescence and (E) Western blot analysis of c-Src and c-Yes kinases in control cells and cells transfected either with a c-Src siRNA or c-Yes siRNA. (F) Quantitative analysis of pY419 SFK at the ZA in control, c-Src KD, and c-Yes KD MCF-7 cells. Data in B, C, and F are means ± SEM for at least 40 contacts per condition. ****, p
    Figure Legend Snippet: c-Src and c-Yes contribute to junctional SFK signaling in MCF-7 cells. (A and B) Quantitative immunofluorescence analysis of NMIIB at the ZA in MCF-7 cells treated with control, PP2, RPTPα siRNA, and RPTPα siRNA+PP2 (25 μM, 1 h). Representative images of E-cadherin and NMIIB in control and PP2-treated cells (A) and quantitative line-scan analysis of NMIIB fluorescence at junctions (B). (C) Normalized initial recoil values for laser nanoscissor experiments performed in MCF-7 cells treated with control, PP2 (25 μM, 1 h) and Y-27632 (30 μM, 1 h). (D) Immunofluorescence and (E) Western blot analysis of c-Src and c-Yes kinases in control cells and cells transfected either with a c-Src siRNA or c-Yes siRNA. (F) Quantitative analysis of pY419 SFK at the ZA in control, c-Src KD, and c-Yes KD MCF-7 cells. Data in B, C, and F are means ± SEM for at least 40 contacts per condition. ****, p

    Techniques Used: Immunofluorescence, Fluorescence, Western Blot, Transfection

    3) Product Images from "The Focal Adhesion: A Regulated Component of Aortic Stiffness"

    Article Title: The Focal Adhesion: A Regulated Component of Aortic Stiffness

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0062461

    LPA stimulation increases FA size in a PP2-sensitive manner. Top: Deconvolution microscopy of FAs. Representative FAs are shown in an expanded view in the insets. All FAs in each cell, not just those featured in the insets, were analyzed to determine the mean FA area. Scale bars: top, 20 µm; bottom, 5 µm. Bottom: Mean FA area is significantly greater in cells stimulated with LPA (n = 82 cells). ***p
    Figure Legend Snippet: LPA stimulation increases FA size in a PP2-sensitive manner. Top: Deconvolution microscopy of FAs. Representative FAs are shown in an expanded view in the insets. All FAs in each cell, not just those featured in the insets, were analyzed to determine the mean FA area. Scale bars: top, 20 µm; bottom, 5 µm. Bottom: Mean FA area is significantly greater in cells stimulated with LPA (n = 82 cells). ***p

    Techniques Used: Microscopy

    Model. (A) Tension-induced, Src- and FAK-mediated growth and remodeling of dynamic focal adhesions in aortic VSMCs leads to cell-matrix adhesion strengthening (cortical stiffening) in response to contractile stimulus. This strengthening is required for adequate force and stiffness transmission from the VSMC to the blood vessel wall. (B) Inhibition of Src with PP2 or FAK with FI-14 inhibits FA dynamics and growth, preventing reinforcement of the cell-matrix linkage. As a result, forces and stiffness generated by the activated VSMC cannot propagate efficiently to the tissue. (C) Inhibition of MLCK with ML-9 reduces contractile force and, as a result, lessens reinforcement of the cell-matrix linkage. Consequently, force and stiffness development in the aortic wall are reduced.
    Figure Legend Snippet: Model. (A) Tension-induced, Src- and FAK-mediated growth and remodeling of dynamic focal adhesions in aortic VSMCs leads to cell-matrix adhesion strengthening (cortical stiffening) in response to contractile stimulus. This strengthening is required for adequate force and stiffness transmission from the VSMC to the blood vessel wall. (B) Inhibition of Src with PP2 or FAK with FI-14 inhibits FA dynamics and growth, preventing reinforcement of the cell-matrix linkage. As a result, forces and stiffness generated by the activated VSMC cannot propagate efficiently to the tissue. (C) Inhibition of MLCK with ML-9 reduces contractile force and, as a result, lessens reinforcement of the cell-matrix linkage. Consequently, force and stiffness development in the aortic wall are reduced.

    Techniques Used: Transmission Assay, Inhibition, Generated

    PE induces FAK and Src-mediated tyrosine phosphorylation of FA proteins in dVSMCs. (A) Typical blot, phosphotyrosine screening of mouse aorta tissue homogenates. PE increases tyrosine phosphorylation, and pre-treatment with Src inhibitor PP2 decreases tyrosine phosphorylation. (B) Mean densitometry of phosphotyrosine bands indicated. (C) Phospho-FAK Y925 increases in response to PE in a PP2-inhibitable manner, mean densitometry (n = 9 mice, 3 experiments). (D-E) Phospho-CAS Y165 and phospho-paxillin Y118 increase in response to PE in a FI-14-inhibitable manner, mean densitometry (n = 9 mice, 3 experiments). The brightness of the representative bands in Insets C-E has been uniformly altered for visual display; however, unaltered images were used for densitometry quantitation. *p
    Figure Legend Snippet: PE induces FAK and Src-mediated tyrosine phosphorylation of FA proteins in dVSMCs. (A) Typical blot, phosphotyrosine screening of mouse aorta tissue homogenates. PE increases tyrosine phosphorylation, and pre-treatment with Src inhibitor PP2 decreases tyrosine phosphorylation. (B) Mean densitometry of phosphotyrosine bands indicated. (C) Phospho-FAK Y925 increases in response to PE in a PP2-inhibitable manner, mean densitometry (n = 9 mice, 3 experiments). (D-E) Phospho-CAS Y165 and phospho-paxillin Y118 increase in response to PE in a FI-14-inhibitable manner, mean densitometry (n = 9 mice, 3 experiments). The brightness of the representative bands in Insets C-E has been uniformly altered for visual display; however, unaltered images were used for densitometry quantitation. *p

    Techniques Used: Mouse Assay, Quantitation Assay

    Aortic tissue stiffening during contractile stimulation is decreased by inhibition of Src, FAK, or MLCK. (A) Tissue stiffness E measured in vitro during PE-induced contraction at optimum length L O with small-amplitude (1%), high frequency (40Hz) sinusoidal stretches Δ L . Box height and width for magnified traces: 0.5 mN, 5 µm, and 0.02 s. Upper Inset : Stiffness calculation. Lower Inset : Fluorescent micrograph of aortic ring in cross-section, used to determine ring thickness for calculation of cross-sectional area A . Green: autofluorescent elastic laminae. Blue: cell nuclei. Scale bar, 100 µm. (B) PE-induced stress is significantly lower when pre-treated with MLCK inhibitor ML-9, confirming the importance of myosin activation and contraction to vascular stiffness (n = 4). PE-induced stress is also significantly reduced when pre-treated with Src inhibitor PP2 and FAK inhibitor 14. (C) PE-induced stiffening is significantly reduced when pre-treated with MLCK inhibitor ML-9, confirming the importance of myosin activation to aortic stiffness. Stiffening is also significantly lower when pre-treated with Src inhibitor PP2 and FAK inhibitor 14, indicating a role for Src, FAK, and FA proteins in aortic stiffness. n = 10 untreated, 6 ML-9, 4 PP2, 5 FI-14 rings. *p
    Figure Legend Snippet: Aortic tissue stiffening during contractile stimulation is decreased by inhibition of Src, FAK, or MLCK. (A) Tissue stiffness E measured in vitro during PE-induced contraction at optimum length L O with small-amplitude (1%), high frequency (40Hz) sinusoidal stretches Δ L . Box height and width for magnified traces: 0.5 mN, 5 µm, and 0.02 s. Upper Inset : Stiffness calculation. Lower Inset : Fluorescent micrograph of aortic ring in cross-section, used to determine ring thickness for calculation of cross-sectional area A . Green: autofluorescent elastic laminae. Blue: cell nuclei. Scale bar, 100 µm. (B) PE-induced stress is significantly lower when pre-treated with MLCK inhibitor ML-9, confirming the importance of myosin activation and contraction to vascular stiffness (n = 4). PE-induced stress is also significantly reduced when pre-treated with Src inhibitor PP2 and FAK inhibitor 14. (C) PE-induced stiffening is significantly reduced when pre-treated with MLCK inhibitor ML-9, confirming the importance of myosin activation to aortic stiffness. Stiffening is also significantly lower when pre-treated with Src inhibitor PP2 and FAK inhibitor 14, indicating a role for Src, FAK, and FA proteins in aortic stiffness. n = 10 untreated, 6 ML-9, 4 PP2, 5 FI-14 rings. *p

    Techniques Used: Inhibition, In Vitro, Activation Assay

    Cortical stiffness measurement in VSMCs. (A) Controlled force pulses generated by magnetic tweezers displace aortic VSMC-adherent, RGD-coated superparamagnetic beads (2.8 µm) to measure the stiffness of the bead-focal adhesion-cortical cytoskeleton linkage (see Methods ). The force F exerted on a bead depends on the induced magnetic moment in the bead m and the spatial gradient of the magnetic field B , which depends critically on the sharpness of the probe’s tip, characterized by its radius of curvature R . (B) Calibration curve relating the force exerted on a bead (d = 2.8 µm) to its distance from the MT tip (R = 40 µm) and the current through the electromagnet solenoid (I = 1.5 A). The gray box denotes the operating range used for the MT experiments, i.e. the distance that is set between the MT tip and a bead before pulling commences. (C) Mean cortical stiffness increases with LPA stimulation in a PP2-attenuated manner. Right: BSA beads, which do not bind integrins but adhere nonspecifically, do not register an increase in stiffness with LPA. *p
    Figure Legend Snippet: Cortical stiffness measurement in VSMCs. (A) Controlled force pulses generated by magnetic tweezers displace aortic VSMC-adherent, RGD-coated superparamagnetic beads (2.8 µm) to measure the stiffness of the bead-focal adhesion-cortical cytoskeleton linkage (see Methods ). The force F exerted on a bead depends on the induced magnetic moment in the bead m and the spatial gradient of the magnetic field B , which depends critically on the sharpness of the probe’s tip, characterized by its radius of curvature R . (B) Calibration curve relating the force exerted on a bead (d = 2.8 µm) to its distance from the MT tip (R = 40 µm) and the current through the electromagnet solenoid (I = 1.5 A). The gray box denotes the operating range used for the MT experiments, i.e. the distance that is set between the MT tip and a bead before pulling commences. (C) Mean cortical stiffness increases with LPA stimulation in a PP2-attenuated manner. Right: BSA beads, which do not bind integrins but adhere nonspecifically, do not register an increase in stiffness with LPA. *p

    Techniques Used: Generated

    4) Product Images from "Semaphorin 4D/Plexin-B1 Induces Endothelial Cell Migration through the Activation of PYK2, Src, and the Phosphatidylinositol 3-Kinase-Akt Pathway"

    Article Title: Semaphorin 4D/Plexin-B1 Induces Endothelial Cell Migration through the Activation of PYK2, Src, and the Phosphatidylinositol 3-Kinase-Akt Pathway

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.25.16.6889-6898.2005

    Blockade of Src prevents Semaphorin 4D-induced Akt activation and cell migration. (A) PP2 blocks Src and Akt phosphorylation in Semaphorin 4D-treated endothelial cells. Endothelial cells were preincubated with either 10 μM PP2 (PP2) or DMSO (C) and treated with 200 ng/ml Semaphorin 4D for the indicated periods of time, lysed, and analyzed for the presence of phosphorylated Akt and Src. Phospho-Src (WB: P-Src) was seen after 1 min of treatment in cells preincubated with DMSO but not in cells grown in PP2 (upper panels). Total Src was used as a loading control (WB: Src). Phosphorylation of Akt is also seen in DMSO-treated control cells but not in PP2 treated populations [WB: P-Akt (Thr 308), lower panels]. Total Akt is used as a loading control (WB: Akt). (B) PP2 blocks Semaphorin 4D/Plexin-B1-mediated chemotaxis. Endothelial cells (control) or cells stably expressing Trk-A/Plexin-B1 chimeric receptors (Trk-A/PB1) were preincubated with either DMSO vehicle control (C) or 10 μM of the Src inhibitor PP2 (PP2) and then used in a cell migration assay with Semaphorin 4D (S4D) or 100 ng/ml NGF (NGF) as the chemoattractants. Media containing 10% fetal bovine serum (S) were used as positive controls for migration. The bars represent the fold increase of migration as determined by densitometry relative to that seen in negative control wells containing 0.1% BSA (C).
    Figure Legend Snippet: Blockade of Src prevents Semaphorin 4D-induced Akt activation and cell migration. (A) PP2 blocks Src and Akt phosphorylation in Semaphorin 4D-treated endothelial cells. Endothelial cells were preincubated with either 10 μM PP2 (PP2) or DMSO (C) and treated with 200 ng/ml Semaphorin 4D for the indicated periods of time, lysed, and analyzed for the presence of phosphorylated Akt and Src. Phospho-Src (WB: P-Src) was seen after 1 min of treatment in cells preincubated with DMSO but not in cells grown in PP2 (upper panels). Total Src was used as a loading control (WB: Src). Phosphorylation of Akt is also seen in DMSO-treated control cells but not in PP2 treated populations [WB: P-Akt (Thr 308), lower panels]. Total Akt is used as a loading control (WB: Akt). (B) PP2 blocks Semaphorin 4D/Plexin-B1-mediated chemotaxis. Endothelial cells (control) or cells stably expressing Trk-A/Plexin-B1 chimeric receptors (Trk-A/PB1) were preincubated with either DMSO vehicle control (C) or 10 μM of the Src inhibitor PP2 (PP2) and then used in a cell migration assay with Semaphorin 4D (S4D) or 100 ng/ml NGF (NGF) as the chemoattractants. Media containing 10% fetal bovine serum (S) were used as positive controls for migration. The bars represent the fold increase of migration as determined by densitometry relative to that seen in negative control wells containing 0.1% BSA (C).

    Techniques Used: Activation Assay, Migration, Western Blot, Chemotaxis Assay, Stable Transfection, Expressing, Cell Migration Assay, Negative Control

    Semaphorin 4D induces the tyrosine phosphorylation of PYK2 and Src in endothelial cells. (A) Treatment of endothelial cells with Semaphorin 4D and cells stably expressing Trk-A/Plexin-B1 chimeric receptor constructs with NGF induces changes in tyrosine phosphorylation. Endothelial cells and cells stably expressing Trk-A/Plexin-B1 chimeric receptors were treated with 200 ng/ml Semaphorin 4D (S4D) or 100 ng/ml NGF (NGF), respectively, for the indicated periods of time, lysed, and analyzed for the presence of tyrosine phosphoproteins (WB: P-Y). Changes in tyrosine phosphorylation of proteins of approximately 110 and 60 kDa are detected. (B) PYK2 is activated in endothelial cells in response to S4D. Endothelial cells were treated with 200 ng/ml Semaphorin 4D for the indicated periods of time, lysed, and immunoblotted with antibodies specific for different sites of tyrosine phosphorylation on PYK2. The levels of phosphorylation on Y579 and 580, residues that reflect kinase activity, and Y881, corresponding to the proposed Grb-2 binding site in the C-terminal domain, are observed starting at 1 min and 3 min after treatment with Semaphorin 4D, respectively. (C) Src is found in tyrosine-phosphorylated molecular complexes in endothelial cells in response to S4D. Cells were treated with 200 ng/ml Semaphorin 4D for the indicated periods of time, lysed, and immunoprecipitated for tyrosine-phosphorylated proteins. The detection of Src in the tyrosine immunoprecipitated fraction dramatically increased at 1 min after treatment (IP: P-Y and WB: Src; upper panel). Total Src was used as a loading control (WB: Src; lower panel). (D) Blockade of Src has no effect on Semaphorin 4D-induced PYK2 activation. Endothelial cells were preincubated with either DMSO vehicle control (C) or 10 μM PP2 (PP2) and treated with 200 ng/ml Semaphorin 4D for the indicated periods of time. Cells were lysed, immunoprecipitated for PYK2 (IP: PYK2), and immunoblotted for the presence of the phosphorylated tyrosine residues Y579 and Y580, located in the activation loop (WB: P-PYK2 Y579/580). Phosphorylated tyrosine residues were detected in the immunoprecipitated fraction starting at approximately 1 min after treatment in both control and PP2 treated populations. Total PYK2 was used as a loading control (WB: PYK2). (E) Blockade of Src prevents Semaphorin 4D-induced phosphorylation of p85 PI3K subunit. Endothelial cells were preincubated with either DMSO vehicle control (C) or 10 μM PP2 (PP2) and treated with 200 ng/ml Semaphorin 4D for the indicated periods of time. Cells were lysed, immunoprecipitated for p85, and immunoblotted for the presence of the phosphorylated tyrosine residues (IP: p85 and WB: P-Y). Phosphorylated tyrosine residues were detected in the immunoprecipitated fraction starting at approximately 1 min after treatment in the DMSO populations but not in cells incubated with PP2 (upper panel). Total p85 was used as a loading control (WB: p85; lower panel). MW, molecular mass.
    Figure Legend Snippet: Semaphorin 4D induces the tyrosine phosphorylation of PYK2 and Src in endothelial cells. (A) Treatment of endothelial cells with Semaphorin 4D and cells stably expressing Trk-A/Plexin-B1 chimeric receptor constructs with NGF induces changes in tyrosine phosphorylation. Endothelial cells and cells stably expressing Trk-A/Plexin-B1 chimeric receptors were treated with 200 ng/ml Semaphorin 4D (S4D) or 100 ng/ml NGF (NGF), respectively, for the indicated periods of time, lysed, and analyzed for the presence of tyrosine phosphoproteins (WB: P-Y). Changes in tyrosine phosphorylation of proteins of approximately 110 and 60 kDa are detected. (B) PYK2 is activated in endothelial cells in response to S4D. Endothelial cells were treated with 200 ng/ml Semaphorin 4D for the indicated periods of time, lysed, and immunoblotted with antibodies specific for different sites of tyrosine phosphorylation on PYK2. The levels of phosphorylation on Y579 and 580, residues that reflect kinase activity, and Y881, corresponding to the proposed Grb-2 binding site in the C-terminal domain, are observed starting at 1 min and 3 min after treatment with Semaphorin 4D, respectively. (C) Src is found in tyrosine-phosphorylated molecular complexes in endothelial cells in response to S4D. Cells were treated with 200 ng/ml Semaphorin 4D for the indicated periods of time, lysed, and immunoprecipitated for tyrosine-phosphorylated proteins. The detection of Src in the tyrosine immunoprecipitated fraction dramatically increased at 1 min after treatment (IP: P-Y and WB: Src; upper panel). Total Src was used as a loading control (WB: Src; lower panel). (D) Blockade of Src has no effect on Semaphorin 4D-induced PYK2 activation. Endothelial cells were preincubated with either DMSO vehicle control (C) or 10 μM PP2 (PP2) and treated with 200 ng/ml Semaphorin 4D for the indicated periods of time. Cells were lysed, immunoprecipitated for PYK2 (IP: PYK2), and immunoblotted for the presence of the phosphorylated tyrosine residues Y579 and Y580, located in the activation loop (WB: P-PYK2 Y579/580). Phosphorylated tyrosine residues were detected in the immunoprecipitated fraction starting at approximately 1 min after treatment in both control and PP2 treated populations. Total PYK2 was used as a loading control (WB: PYK2). (E) Blockade of Src prevents Semaphorin 4D-induced phosphorylation of p85 PI3K subunit. Endothelial cells were preincubated with either DMSO vehicle control (C) or 10 μM PP2 (PP2) and treated with 200 ng/ml Semaphorin 4D for the indicated periods of time. Cells were lysed, immunoprecipitated for p85, and immunoblotted for the presence of the phosphorylated tyrosine residues (IP: p85 and WB: P-Y). Phosphorylated tyrosine residues were detected in the immunoprecipitated fraction starting at approximately 1 min after treatment in the DMSO populations but not in cells incubated with PP2 (upper panel). Total p85 was used as a loading control (WB: p85; lower panel). MW, molecular mass.

    Techniques Used: Stable Transfection, Expressing, Construct, Western Blot, Activity Assay, Binding Assay, Immunoprecipitation, Activation Assay, Incubation

    Plexin-B1 activation promotes the assembly of multimeric signaling complexes. (A) Plexin-B1 is tyrosine phosphorylated upon activation. Lysates of endothelial cells stably expressing myc-tagged Trk-A/Plexin-B1 chimeric receptors treated with 100 ng/ml NGF for the indicated periods of time were immunoprecipitated for the receptor (IP: myc) and immunoblotted for the presence of phosphorylated tyrosine residues (WB: P-Y). A phosphotyrosine band of the appropriate size begins to appear in the immunoprecipitated fraction at about 1 min (upper panel). Total receptor (WB: myc) was used as a loading control (lower panel). (B) 293T cells were cotransfected with myc-tagged Trk-A/Plexin-B1 and either DN-PYK2 (DN-PYK2), control DNA (C), or control DNA preceded by incubation with the Src inhibitor PP2 (PP2), followed by treatment with 100 ng/ml NGF. An immunoblot for myc was performed on phosphotyrosine-immunoprecipitated fractions (IP: P-Y and WB: myc). An increase in receptor phosphorylation is seen in response to NGF in control cells (lane 2) and cells pretreated with PP2 (lane 4) but not in cells expressing DN-PYK2 (lane 6). (C) PYK2, p85, and Src bind Plexin-B1 upon receptor activation. Lysates of endothelial cells stably expressing myc-tagged Trk-A/Plexin-B1 chimeric receptors treated with 100 ng/ml NGF for the indicated periods of time were immunoprecipitated for the receptor (IP: myc) and immunoblotted for the presence of PYK2, p85, and Src. Each of these proteins is seen associating with the immunoprecipitated receptor complex at 1 min following treatment. Total PYK2, p85, Src, and myc immunoblots were used as loading controls. As a negative control, a hemagglutinin immunoprecipitation was performed (IP: HA; right panels) which showed no proteins after 0 or 5 min treatment with NGF when probed for PYK2, p85, Src, and myc. Total PYK2, p85, Src, and myc were used as loading controls (WB lanes; lower panels).
    Figure Legend Snippet: Plexin-B1 activation promotes the assembly of multimeric signaling complexes. (A) Plexin-B1 is tyrosine phosphorylated upon activation. Lysates of endothelial cells stably expressing myc-tagged Trk-A/Plexin-B1 chimeric receptors treated with 100 ng/ml NGF for the indicated periods of time were immunoprecipitated for the receptor (IP: myc) and immunoblotted for the presence of phosphorylated tyrosine residues (WB: P-Y). A phosphotyrosine band of the appropriate size begins to appear in the immunoprecipitated fraction at about 1 min (upper panel). Total receptor (WB: myc) was used as a loading control (lower panel). (B) 293T cells were cotransfected with myc-tagged Trk-A/Plexin-B1 and either DN-PYK2 (DN-PYK2), control DNA (C), or control DNA preceded by incubation with the Src inhibitor PP2 (PP2), followed by treatment with 100 ng/ml NGF. An immunoblot for myc was performed on phosphotyrosine-immunoprecipitated fractions (IP: P-Y and WB: myc). An increase in receptor phosphorylation is seen in response to NGF in control cells (lane 2) and cells pretreated with PP2 (lane 4) but not in cells expressing DN-PYK2 (lane 6). (C) PYK2, p85, and Src bind Plexin-B1 upon receptor activation. Lysates of endothelial cells stably expressing myc-tagged Trk-A/Plexin-B1 chimeric receptors treated with 100 ng/ml NGF for the indicated periods of time were immunoprecipitated for the receptor (IP: myc) and immunoblotted for the presence of PYK2, p85, and Src. Each of these proteins is seen associating with the immunoprecipitated receptor complex at 1 min following treatment. Total PYK2, p85, Src, and myc immunoblots were used as loading controls. As a negative control, a hemagglutinin immunoprecipitation was performed (IP: HA; right panels) which showed no proteins after 0 or 5 min treatment with NGF when probed for PYK2, p85, Src, and myc. Total PYK2, p85, Src, and myc were used as loading controls (WB lanes; lower panels).

    Techniques Used: Activation Assay, Stable Transfection, Expressing, Immunoprecipitation, Western Blot, Incubation, Negative Control

    5) Product Images from "Identification of a functional interaction between Kv4.3 channels and c-Src tyrosine kinase"

    Article Title: Identification of a functional interaction between Kv4.3 channels and c-Src tyrosine kinase

    Journal:

    doi: 10.1016/j.bbamcr.2008.06.011

    PP2, a selective inhibitor of Src family tyrosine kinases, reduces Kv4.3 currents
    Figure Legend Snippet: PP2, a selective inhibitor of Src family tyrosine kinases, reduces Kv4.3 currents

    Techniques Used:

    6) Product Images from "HMBPP analog prodrugs bypass energy-dependent uptake to promote efficient BTN3A1-mediated malignant cell lysis by Vγ9Vδ2 T lymphocyte effectors"

    Article Title: HMBPP analog prodrugs bypass energy-dependent uptake to promote efficient BTN3A1-mediated malignant cell lysis by Vγ9Vδ2 T lymphocyte effectors

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1501833

    Target cell lysis by Vγ9Vδ2 T effectors requires activity of Src kinases and is enhanced by POM 2 -C-HMBP during short-term pre-treatment A) Target and effectors cells were mixed and simultaneously exposed to 100 nM HMBPP with or without 10 μM PP2 for four hours. B) Quantification of panel A. C) Effector T cells demonstrate autolysis in the continued presence of HMBPP. D) K562 cell population gating scheme for 2 hour pre-treatment of K562 cells with phosphorus compounds (100 nM), followed by 4 hour incubation with effector cells. E) Dose response quantification of 2 hour pre-treatment lysis assays for both HMBPP and POM 2 -C-HMBP with EC 50 values. F) Dose response quantification of IFNγ secretion following a 2 hour phosphorus compound pre-treatment and 24 hour incubation with effector cells. Flow plots are representative of greater than three independent experiments. *, significantly different from control; # significantly different from treatment.
    Figure Legend Snippet: Target cell lysis by Vγ9Vδ2 T effectors requires activity of Src kinases and is enhanced by POM 2 -C-HMBP during short-term pre-treatment A) Target and effectors cells were mixed and simultaneously exposed to 100 nM HMBPP with or without 10 μM PP2 for four hours. B) Quantification of panel A. C) Effector T cells demonstrate autolysis in the continued presence of HMBPP. D) K562 cell population gating scheme for 2 hour pre-treatment of K562 cells with phosphorus compounds (100 nM), followed by 4 hour incubation with effector cells. E) Dose response quantification of 2 hour pre-treatment lysis assays for both HMBPP and POM 2 -C-HMBP with EC 50 values. F) Dose response quantification of IFNγ secretion following a 2 hour phosphorus compound pre-treatment and 24 hour incubation with effector cells. Flow plots are representative of greater than three independent experiments. *, significantly different from control; # significantly different from treatment.

    Techniques Used: Lysis, Activity Assay, Incubation, Flow Cytometry

    7) Product Images from "Metalloproteinase/Presenilin1 processing of ephrinB regulates EphB-induced Src phosphorylation and signaling"

    Article Title: Metalloproteinase/Presenilin1 processing of ephrinB regulates EphB-induced Src phosphorylation and signaling

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7601031

    ( A ) EphrinB2/CTF2 promotes ephrinB phosphorylation in an Src-dependent manner. C6 rat glioma cells were transfected as follows: vector (lane 1), ephrinB2/CTF2-myc3 alone (lanes 2, 5, and 6), N-cad/CTF2 (lane 3) or ephrinB2/CTF2-myc3, and dominant-negative (DN) Src (lane 4) as indicated. At 24 h following transfection, cells in samples 5 and 6 were treated with 1 μM SU6656 or 10 μM PP2, respectively, for 1 h. Extracts in Triton X-100 extraction buffer were IPed with antibody C18 and obtained IPs were probed with anti-phosphotyrosine pY99 (p-Tyr, upper panel) or C-18 (ephrinB-FL, second panel). The three lower panels indicate levels of transfected proteins (input). ( B ) EphrinB2/CTF2 inhibits γ-secretase processing of ephrinB2 in an Src-dependent manner. PS1+/+ cells were transduced with pMX-ephrinB2 and then transiently transfected with N-cad/CTF2 (lane 1), vector alone (lane 2), ephrinB2/CTF2-myc3 (lanes 3–5), or cotransfected with ephrinB2/CTF2-myc3 and dominant-negative Src (DN-Src, lane 6). At 24 h following transfection, all cultures were made 10 μM in lactacystin and then incubated for an additional 10 h before harvesting. Cultures 4 and 5 were treated with 1 μM SU6656 (lane 4) or 10 μM PP2 (lane 5) 1 h prior to harvesting. Cell lysates were probed for endogenous ephrinB2/CTF2 using C18 (panel 3). Expression of exogenous protein is indicated in panels 4–6. Arrowheads indicate ephrinB2 (ephrinB2-FL), ephrinB2/CTF1, endogenous ephrinB2/CTF2, exogenous ephrinB2/CTF2 (ephrinB2/CTF2-myc3), N-cadherin/CTF2 (N-cad/CTF2) and Src.
    Figure Legend Snippet: ( A ) EphrinB2/CTF2 promotes ephrinB phosphorylation in an Src-dependent manner. C6 rat glioma cells were transfected as follows: vector (lane 1), ephrinB2/CTF2-myc3 alone (lanes 2, 5, and 6), N-cad/CTF2 (lane 3) or ephrinB2/CTF2-myc3, and dominant-negative (DN) Src (lane 4) as indicated. At 24 h following transfection, cells in samples 5 and 6 were treated with 1 μM SU6656 or 10 μM PP2, respectively, for 1 h. Extracts in Triton X-100 extraction buffer were IPed with antibody C18 and obtained IPs were probed with anti-phosphotyrosine pY99 (p-Tyr, upper panel) or C-18 (ephrinB-FL, second panel). The three lower panels indicate levels of transfected proteins (input). ( B ) EphrinB2/CTF2 inhibits γ-secretase processing of ephrinB2 in an Src-dependent manner. PS1+/+ cells were transduced with pMX-ephrinB2 and then transiently transfected with N-cad/CTF2 (lane 1), vector alone (lane 2), ephrinB2/CTF2-myc3 (lanes 3–5), or cotransfected with ephrinB2/CTF2-myc3 and dominant-negative Src (DN-Src, lane 6). At 24 h following transfection, all cultures were made 10 μM in lactacystin and then incubated for an additional 10 h before harvesting. Cultures 4 and 5 were treated with 1 μM SU6656 (lane 4) or 10 μM PP2 (lane 5) 1 h prior to harvesting. Cell lysates were probed for endogenous ephrinB2/CTF2 using C18 (panel 3). Expression of exogenous protein is indicated in panels 4–6. Arrowheads indicate ephrinB2 (ephrinB2-FL), ephrinB2/CTF1, endogenous ephrinB2/CTF2, exogenous ephrinB2/CTF2 (ephrinB2/CTF2-myc3), N-cadherin/CTF2 (N-cad/CTF2) and Src.

    Techniques Used: Transfection, Plasmid Preparation, Dominant Negative Mutation, Transduction, Incubation, Expressing

    8) Product Images from "Angiomotin-like2 Gene (amotl2) Is Required for Migration and Proliferation of Endothelial Cells during Angiogenesis *"

    Article Title: Angiomotin-like2 Gene (amotl2) Is Required for Migration and Proliferation of Endothelial Cells during Angiogenesis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.296806

    Amotl2 promotes MAPK activation through c-Src depending on its Tyr-103 phosphorylation. A , Amotl2-mediated MAPK activation depended on Src kinase activity in HEK293T cells. Addition of the Src kinase inhibitor PP2 inhibited Myc-AMOTL2-up-regulated p-ERK1/2
    Figure Legend Snippet: Amotl2 promotes MAPK activation through c-Src depending on its Tyr-103 phosphorylation. A , Amotl2-mediated MAPK activation depended on Src kinase activity in HEK293T cells. Addition of the Src kinase inhibitor PP2 inhibited Myc-AMOTL2-up-regulated p-ERK1/2

    Techniques Used: Activation Assay, Activity Assay

    9) Product Images from "Integrin α6β4-Src-AKT signaling induces cellular senescence by counteracting apoptosis in irradiated tumor cells and tissues"

    Article Title: Integrin α6β4-Src-AKT signaling induces cellular senescence by counteracting apoptosis in irradiated tumor cells and tissues

    Journal: Cell Death and Differentiation

    doi: 10.1038/s41418-018-0114-7

    Integrin α6β4-Src-AKT signaling is critical for IR-induced cellular senescence in the tumor tissues of xenograft mice . H460 cells (1 × 10 6 ) were injected subcutaneously into female nude mice. When the tumor volume reached 100 mm 3 , 200 μl AteloGene gel containing integrin β4 Si or PP2 (a Src inhibitor) was injected to encompass the whole tumor mass. After 1 day, 12 Gy radiation was applied. a The tumor size (mm 3 ) was measured at the indicated times. Error bars represent ± SEM ( n = 5). b , c H E staining, TUNEL, Ki-67 immunostaining, and p21 immunostaining were performed on tumor sections derived from xenograft mice exposed to IR ( b ), and the Ki-67-, p21-, and TUNEL-positive cells per field were quantified ( c ). Scale bars indicate 10 μm. Arrows indicate apoptotic cells. d Tumor lysates obtained at 8 days after irradiation were subjected to immunoblot analysis. e Pictures of SA-β-Gal-stained tumors 8 days after irradiation ( f-j ). f Possible role of the ITGα6β4-Src-AKT signaling pathway in the IR-induced premature senescence. k Possible role of the ITGα6β4-Src-AKT signaling pathway in the IR-induced premature senescence
    Figure Legend Snippet: Integrin α6β4-Src-AKT signaling is critical for IR-induced cellular senescence in the tumor tissues of xenograft mice . H460 cells (1 × 10 6 ) were injected subcutaneously into female nude mice. When the tumor volume reached 100 mm 3 , 200 μl AteloGene gel containing integrin β4 Si or PP2 (a Src inhibitor) was injected to encompass the whole tumor mass. After 1 day, 12 Gy radiation was applied. a The tumor size (mm 3 ) was measured at the indicated times. Error bars represent ± SEM ( n = 5). b , c H E staining, TUNEL, Ki-67 immunostaining, and p21 immunostaining were performed on tumor sections derived from xenograft mice exposed to IR ( b ), and the Ki-67-, p21-, and TUNEL-positive cells per field were quantified ( c ). Scale bars indicate 10 μm. Arrows indicate apoptotic cells. d Tumor lysates obtained at 8 days after irradiation were subjected to immunoblot analysis. e Pictures of SA-β-Gal-stained tumors 8 days after irradiation ( f-j ). f Possible role of the ITGα6β4-Src-AKT signaling pathway in the IR-induced premature senescence. k Possible role of the ITGα6β4-Src-AKT signaling pathway in the IR-induced premature senescence

    Techniques Used: Mouse Assay, Injection, Staining, TUNEL Assay, Immunostaining, Derivative Assay, Irradiation

    10) Product Images from "Phospholipase C?2 is required for basal but not oestrogen deficiency-induced bone resorption"

    Article Title: Phospholipase C?2 is required for basal but not oestrogen deficiency-induced bone resorption

    Journal: European Journal of Clinical Investigation

    doi: 10.1111/j.1365-2362.2011.02556.x

    Cellular adhesion triggers PLCγ2 phosphorylation. (a) Expression of PLCγ2 in macrophages (MΦ) and osteoclasts (OC). Wild-type (WT) and PLCγ2 −/− bone marrow cells were cultured in the presence of 50 ng/mL M-CSF with (OC) or without (MΦ) 50 ng/mL RANKL for 4 days, followed by preparation of whole-cell lysates (WCL) and immunoblotting for PLCγ2 and β-actin. B-C, Stimulus-induced phosphorylation of PLCγ2. Wild-type macrophages were treated with 50 ng/mL M-CSF, 50 ng/mL RANKL or kept unstimulated in suspension, or they were plated on tissue culture-treated plastic dishes (Adh). After 30 min of incubation, cell was lysed and processed for immunoprecipitation (IP) of PLCγ2 followed by immunoblotting using anti-phosphotyrosine (PY) antibodies, (b) or WCL were immunoblotted using phosphorylation-specific antibodies against ERK, the p38 MAP kinase (p38) and PLCγ2 or nonphospho-specific antibodies against IκBα (c). Immunoblotting for ERK, p38 and PLCγ2 served as loading control. (d) Role of Src-family kinases in PLCγ2 phosphorylation. Wild-type macrophages were pretreated in the presence or absence of 10 μM PP2 and then stimulated and their PLCγ2 phosphorylation tested as in panel b. Results shown represent 3–5 independent experiments with similar results.
    Figure Legend Snippet: Cellular adhesion triggers PLCγ2 phosphorylation. (a) Expression of PLCγ2 in macrophages (MΦ) and osteoclasts (OC). Wild-type (WT) and PLCγ2 −/− bone marrow cells were cultured in the presence of 50 ng/mL M-CSF with (OC) or without (MΦ) 50 ng/mL RANKL for 4 days, followed by preparation of whole-cell lysates (WCL) and immunoblotting for PLCγ2 and β-actin. B-C, Stimulus-induced phosphorylation of PLCγ2. Wild-type macrophages were treated with 50 ng/mL M-CSF, 50 ng/mL RANKL or kept unstimulated in suspension, or they were plated on tissue culture-treated plastic dishes (Adh). After 30 min of incubation, cell was lysed and processed for immunoprecipitation (IP) of PLCγ2 followed by immunoblotting using anti-phosphotyrosine (PY) antibodies, (b) or WCL were immunoblotted using phosphorylation-specific antibodies against ERK, the p38 MAP kinase (p38) and PLCγ2 or nonphospho-specific antibodies against IκBα (c). Immunoblotting for ERK, p38 and PLCγ2 served as loading control. (d) Role of Src-family kinases in PLCγ2 phosphorylation. Wild-type macrophages were pretreated in the presence or absence of 10 μM PP2 and then stimulated and their PLCγ2 phosphorylation tested as in panel b. Results shown represent 3–5 independent experiments with similar results.

    Techniques Used: Expressing, Cell Culture, Incubation, Immunoprecipitation

    11) Product Images from "RANKL/RANK pathway abrogates cetuximab sensitivity in gastric cancer cells via activation of EGFR and c-Src"

    Article Title: RANKL/RANK pathway abrogates cetuximab sensitivity in gastric cancer cells via activation of EGFR and c-Src

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S110918

    The effect of dasatinib on RANKL-induced cetuximab resistance in SGC-7901 cells. Notes: ( A and B ) SGC-7901 and MGC-803 cells were treated with 100 nM dasatinib or 10 μM PP2 for the indicated times. Expression of c-Src, EGFR, AKT, and ERK and phosphorylation levels were analyzed by Western blot. ( C ) SGC-7901 cells were treated with 100 nM dasatinib for 24 hours, followed by 1 μg/mL sRANKL for 1 hour and then 10 μg/mL cetuximab for 48 hours. The cell viability was assessed by MTT assay. *Comparisons between the cetuximab-treated cells and the combined-treated cells in the control arm, P
    Figure Legend Snippet: The effect of dasatinib on RANKL-induced cetuximab resistance in SGC-7901 cells. Notes: ( A and B ) SGC-7901 and MGC-803 cells were treated with 100 nM dasatinib or 10 μM PP2 for the indicated times. Expression of c-Src, EGFR, AKT, and ERK and phosphorylation levels were analyzed by Western blot. ( C ) SGC-7901 cells were treated with 100 nM dasatinib for 24 hours, followed by 1 μg/mL sRANKL for 1 hour and then 10 μg/mL cetuximab for 48 hours. The cell viability was assessed by MTT assay. *Comparisons between the cetuximab-treated cells and the combined-treated cells in the control arm, P

    Techniques Used: Expressing, Western Blot, MTT Assay

    12) Product Images from "Epidermal Growth Factor Receptor Transactivation Is Required for Mitogen-Activated Protein Kinase Activation by Muscarinic Acetylcholine Receptors in HaCaT Keratinocytes"

    Article Title: Epidermal Growth Factor Receptor Transactivation Is Required for Mitogen-Activated Protein Kinase Activation by Muscarinic Acetylcholine Receptors in HaCaT Keratinocytes

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms151121433

    Src family kinases and PI3K are involved in cholinergic Akt but not ERK activation. ( A , D , G ) Serum-starved HaCaT cells were pretreated with the Src family specific inhibitor PP2 and the non-inhibiting PP3 (both 10 µM) for 10 min ( A ), or with 20 µM PI3K inhibitor Ly 294002 or DMSO for 10 min ( D ), or with 200 nM BIM I, a PKC inhibitor, or DMSO for 20 min ( G ). The cells were then stimulated with 1 mM CCh or 16 nM EGF for 30 min in the presence of the inhibitors. Equal amounts of cell lysates were separated by SDS-PAGE and immunoblotted; ( B , C , E , F , H , I ) The amount of pAkt and pERK was determined by densitometric quantification and normalized to total Akt or ERK. Data are shown relative to the EGF stimulated controls. Bars represent the mean ± SD of at least three independent experiments. Statistical analysis was performed with two-way ANOVA; * p
    Figure Legend Snippet: Src family kinases and PI3K are involved in cholinergic Akt but not ERK activation. ( A , D , G ) Serum-starved HaCaT cells were pretreated with the Src family specific inhibitor PP2 and the non-inhibiting PP3 (both 10 µM) for 10 min ( A ), or with 20 µM PI3K inhibitor Ly 294002 or DMSO for 10 min ( D ), or with 200 nM BIM I, a PKC inhibitor, or DMSO for 20 min ( G ). The cells were then stimulated with 1 mM CCh or 16 nM EGF for 30 min in the presence of the inhibitors. Equal amounts of cell lysates were separated by SDS-PAGE and immunoblotted; ( B , C , E , F , H , I ) The amount of pAkt and pERK was determined by densitometric quantification and normalized to total Akt or ERK. Data are shown relative to the EGF stimulated controls. Bars represent the mean ± SD of at least three independent experiments. Statistical analysis was performed with two-way ANOVA; * p

    Techniques Used: Activation Assay, SDS Page

    13) Product Images from "UVB-mediated down-regulation of proteasome in cultured human primary pterygium fibroblasts"

    Article Title: UVB-mediated down-regulation of proteasome in cultured human primary pterygium fibroblasts

    Journal: BMC Ophthalmology

    doi: 10.1186/s12886-018-0987-8

    Representative western blottings of lysates of pterygium ( a ) and normal conjunctival ( b ) fibroblasts, irradiated with different doses of UVB radiation (0–50 mJ/cm 2 ) after pretreatment with PP2, using antibodies against PSMB5 or α-tubulin
    Figure Legend Snippet: Representative western blottings of lysates of pterygium ( a ) and normal conjunctival ( b ) fibroblasts, irradiated with different doses of UVB radiation (0–50 mJ/cm 2 ) after pretreatment with PP2, using antibodies against PSMB5 or α-tubulin

    Techniques Used: Western Blot, Irradiation

    RT-PCR analysis of PSMB5 ( a ) and Nrf2 ( b ), and qPCR analysis ( c ) of mRNA steady-state levels using total RNA from pterygium and normal conjunctival fibroblasts, respectively, irradiated with different doses of UVB radiation (0–50 mJ/cm 2 ), after pretreatment with PP2. The products for PSMB5 (550 bp), Nrf2 (173 bp) and GAPDH (263 bp) are indicated. L: DNA ladder. (*) indicates significant difference ( p
    Figure Legend Snippet: RT-PCR analysis of PSMB5 ( a ) and Nrf2 ( b ), and qPCR analysis ( c ) of mRNA steady-state levels using total RNA from pterygium and normal conjunctival fibroblasts, respectively, irradiated with different doses of UVB radiation (0–50 mJ/cm 2 ), after pretreatment with PP2. The products for PSMB5 (550 bp), Nrf2 (173 bp) and GAPDH (263 bp) are indicated. L: DNA ladder. (*) indicates significant difference ( p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Irradiation

    14) Product Images from "The Src Family Kinase c-Yes Is Required for Maturation of West Nile Virus Particles"

    Article Title: The Src Family Kinase c-Yes Is Required for Maturation of West Nile Virus Particles

    Journal: Journal of Virology

    doi: 10.1128/JVI.79.18.11943-11951.2005

    WNV RNA levels in PP2-treated SK-N-MC and Vero cells. (A) SK-N-MC cells were infected at a multiplicity of 10 and treated with 20 μM PP2 or DMSO. RNA was harvested at the indicated times postinfection and quantified using quantitative real-time PCR. RNA quantities are expressed as viral genomes/μg total cellular RNA. (B) Vero cells were infected at a multiplicity of 5 and treated with 20 μM PP2 (squares) or DMSO (diamonds). RNA was harvested and quantified as above. In both panels, values represent the averages of three infections.
    Figure Legend Snippet: WNV RNA levels in PP2-treated SK-N-MC and Vero cells. (A) SK-N-MC cells were infected at a multiplicity of 10 and treated with 20 μM PP2 or DMSO. RNA was harvested at the indicated times postinfection and quantified using quantitative real-time PCR. RNA quantities are expressed as viral genomes/μg total cellular RNA. (B) Vero cells were infected at a multiplicity of 5 and treated with 20 μM PP2 (squares) or DMSO (diamonds). RNA was harvested and quantified as above. In both panels, values represent the averages of three infections.

    Techniques Used: Infection, Real-time Polymerase Chain Reaction

    Effect of the SFK inhibitor PP2 on WNV growth. WNV-infected SK-N-MC (A and B) or HEK 293 (C and D) cells were treated with DMSO (diamonds) or 20 μM PP2 (squares). At the indicated times postinfection, supernatant (A and C) and cell-associated virus (B and D) were quantitated by plaque assay on Vero cells. Results shown are the averages of three independent infections. Error bars represent standard deviations.
    Figure Legend Snippet: Effect of the SFK inhibitor PP2 on WNV growth. WNV-infected SK-N-MC (A and B) or HEK 293 (C and D) cells were treated with DMSO (diamonds) or 20 μM PP2 (squares). At the indicated times postinfection, supernatant (A and C) and cell-associated virus (B and D) were quantitated by plaque assay on Vero cells. Results shown are the averages of three independent infections. Error bars represent standard deviations.

    Techniques Used: Infection, Plaque Assay

    WNV E protein maturation is impaired in PP2-treated cells. Vero cells were infected with WNV at a multiplicity of 5 and treated with 10 μM PP2 (or DMSO as a control) 1 h postinfection. Cells were lysed at 24 h postinfection. Lysates were denatured and treated with endoH (H lanes) or PNGase F (F lanes). Samples were resolved by SDS-PAGE, and WNV E and MHC I were detected by Western blotting.
    Figure Legend Snippet: WNV E protein maturation is impaired in PP2-treated cells. Vero cells were infected with WNV at a multiplicity of 5 and treated with 10 μM PP2 (or DMSO as a control) 1 h postinfection. Cells were lysed at 24 h postinfection. Lysates were denatured and treated with endoH (H lanes) or PNGase F (F lanes). Samples were resolved by SDS-PAGE, and WNV E and MHC I were detected by Western blotting.

    Techniques Used: Infection, SDS Page, Western Blot

    ER-associated particles are found in increased numbers in PP2-treated cells. Mock-infected (A and B) or WNV-infected (C, D, and E) Vero cells treated with DMSO (A and C) or 10 μM PP2 (B, D, and E) were processed for electron microscopy at 24 h postinfection. Representative ER-localized virions are indicated by arrows. Bars, 500 nm (A to D) or 100 nm (E). (F) Quantitation of total or ER-localized virus particles per field (13.125 μm 2 ) ± standard error of the mean. Totals represent the average of 14 fields each. Light gray bars, DMSO-treated cells; dark gray bars, PP2-treated cells.
    Figure Legend Snippet: ER-associated particles are found in increased numbers in PP2-treated cells. Mock-infected (A and B) or WNV-infected (C, D, and E) Vero cells treated with DMSO (A and C) or 10 μM PP2 (B, D, and E) were processed for electron microscopy at 24 h postinfection. Representative ER-localized virions are indicated by arrows. Bars, 500 nm (A to D) or 100 nm (E). (F) Quantitation of total or ER-localized virus particles per field (13.125 μm 2 ) ± standard error of the mean. Totals represent the average of 14 fields each. Light gray bars, DMSO-treated cells; dark gray bars, PP2-treated cells.

    Techniques Used: Infection, Electron Microscopy, Quantitation Assay

    PP2 treatment does not result in significant cellular toxicity through 48 h. (A) SK-N-MC cells were treated with the indicated concentrations of PP2 for 48 h. Viable cells were counted using trypan blue exclusion. Cell number is expressed relative to the number of viable cells in DMSO controls. Numbers represent the averages of three wells for each condition. (B) Vero cells were treated with the indicated concentrations of PP2 for 48 h. Numbers of metabolically active cells were quantified by measuring conversion of XTT tetrazolium salt to a formazan dye. Cell number is expressed relative to the number of viable cells in DMSO controls. Numbers represent the averages of three wells for each condition.
    Figure Legend Snippet: PP2 treatment does not result in significant cellular toxicity through 48 h. (A) SK-N-MC cells were treated with the indicated concentrations of PP2 for 48 h. Viable cells were counted using trypan blue exclusion. Cell number is expressed relative to the number of viable cells in DMSO controls. Numbers represent the averages of three wells for each condition. (B) Vero cells were treated with the indicated concentrations of PP2 for 48 h. Numbers of metabolically active cells were quantified by measuring conversion of XTT tetrazolium salt to a formazan dye. Cell number is expressed relative to the number of viable cells in DMSO controls. Numbers represent the averages of three wells for each condition.

    Techniques Used: Metabolic Labelling

    TCID 50 determination of WNV and HSV in the presence of SFK inhibitors. (A) TCID 50 s of WNV on Vero cells in the presence of the indicated concentrations of PP2, PP3, or DMSO. (B) TCID 50 s of Vero cells infected with HSV in the presence of PP2 or DMSO. Results shown are the averages of three independent infections. Error bars represent standard deviations.
    Figure Legend Snippet: TCID 50 determination of WNV and HSV in the presence of SFK inhibitors. (A) TCID 50 s of WNV on Vero cells in the presence of the indicated concentrations of PP2, PP3, or DMSO. (B) TCID 50 s of Vero cells infected with HSV in the presence of PP2 or DMSO. Results shown are the averages of three independent infections. Error bars represent standard deviations.

    Techniques Used: Infection

    PP2 does not inhibit WNV protein synthesis but prevents release of viral particles. (A) WNV E protein was immunoprecipitated from 35 S-labeled lysates from mock-treated, WNV-infected and DMSO-treated, or WNV-infected and PP2 (20 μM)-treated cells. Arrows indicate the expected sizes of the E and prM proteins. In lane 4, an infected cell lysate was subjected to the immunoprecipitation procedure without E-specific antibody. (B) Culture supernatants from 35 S-labeled mock-infected, WNV-infected and DMSO-treated, or WNV-infected and PP2 (20 μM)-treated cells were centrifuged through a 20% sucrose cushion, and pelleted virus was analyzed by SDS-PAGE.
    Figure Legend Snippet: PP2 does not inhibit WNV protein synthesis but prevents release of viral particles. (A) WNV E protein was immunoprecipitated from 35 S-labeled lysates from mock-treated, WNV-infected and DMSO-treated, or WNV-infected and PP2 (20 μM)-treated cells. Arrows indicate the expected sizes of the E and prM proteins. In lane 4, an infected cell lysate was subjected to the immunoprecipitation procedure without E-specific antibody. (B) Culture supernatants from 35 S-labeled mock-infected, WNV-infected and DMSO-treated, or WNV-infected and PP2 (20 μM)-treated cells were centrifuged through a 20% sucrose cushion, and pelleted virus was analyzed by SDS-PAGE.

    Techniques Used: Immunoprecipitation, Labeling, Infection, SDS Page

    15) Product Images from "Quiescence and ?H2AX in neuroblastoma are regulated by ouabain/Na,K-ATPase"

    Article Title: Quiescence and ?H2AX in neuroblastoma are regulated by ouabain/Na,K-ATPase

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2012.159

    Cellular quiescence induced by ouabain in neuroblastoma is mediated by an alternative signalling pathway. ( A ) Immunostaining of BrdU (green) in SH-SY5Y cells treated with 50 nℳ ouabain together with L-type Ca 2+ channel blocker nifedipine (Nif) or CaM kinase inhibitor KN93, respectively. ( B ) Quantification of BrdU-positive cells treated with 50 nℳ ouabain together with nifedipine or KN93, respectively. Pooled results from three randomly selected fields-of-view from three cultures are shown. ( C ) Effect on cell cycle regulators pRb, Rb, cyclins D3, E, CDK1, 2, 4, and p21 in cells treated with 50 nℳ ouabain for 2 days plus nifedipine, KN93, CaM kinase kinase inhibitor STO-609 (STO) or calmodulin inhibitor W-13, plus ( D ) Src inhibitor PP2, MEK/MAPK inhibitor U0126, PKC inhibitor GF109203X (GF), or PKA inhibitor H89. β -actin was used as a loading control. ( E ) Western blot of phosphorylated Akt (pAkt) and total Akt in cells treated with 50 nℳ ouabain for 2 days. * P
    Figure Legend Snippet: Cellular quiescence induced by ouabain in neuroblastoma is mediated by an alternative signalling pathway. ( A ) Immunostaining of BrdU (green) in SH-SY5Y cells treated with 50 nℳ ouabain together with L-type Ca 2+ channel blocker nifedipine (Nif) or CaM kinase inhibitor KN93, respectively. ( B ) Quantification of BrdU-positive cells treated with 50 nℳ ouabain together with nifedipine or KN93, respectively. Pooled results from three randomly selected fields-of-view from three cultures are shown. ( C ) Effect on cell cycle regulators pRb, Rb, cyclins D3, E, CDK1, 2, 4, and p21 in cells treated with 50 nℳ ouabain for 2 days plus nifedipine, KN93, CaM kinase kinase inhibitor STO-609 (STO) or calmodulin inhibitor W-13, plus ( D ) Src inhibitor PP2, MEK/MAPK inhibitor U0126, PKC inhibitor GF109203X (GF), or PKA inhibitor H89. β -actin was used as a loading control. ( E ) Western blot of phosphorylated Akt (pAkt) and total Akt in cells treated with 50 nℳ ouabain for 2 days. * P

    Techniques Used: Immunostaining, Chick Chorioallantoic Membrane Assay, Western Blot

    16) Product Images from "Transactivation of epidermal growth factor receptor through platelet-activating factor/receptor in ovarian cancer cells"

    Article Title: Transactivation of epidermal growth factor receptor through platelet-activating factor/receptor in ovarian cancer cells

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-014-0085-6

    Effect of Src and MMP inhibitors on EGFR phosphorylation. (A) SKOV-3 cells were treated with 100 nM PAF for 0, 5, 10, 15, 20, or 25 min. Following PAF treatment, cells were lysed and lysates were evaluated by Western blotting. Data were normalized to total Src protein expression and are expressed as the fold change (average ± S.E.M.) in phospho-Src compared to vehicle-treated cells. Representative blots for phosphor/total-EGFR are shown. (B) SKOV-3 cells were treated with the Src inhibitor PP2 (20 μM) for 1 hour before stimulation with PAF (100 nM) or EGF (5 ng/ml) for 5 min. Cells were harvested and subjected to Western blot. (C) SKOV-3 cells were pretreated for 1 h with the metalloproteinase inhibitor GM6001 (20 μM). Cells were then stimulated with either PAF (100 nM) or EGF (5 ng/ml) for 5 min before they were harvested and immunoblotting. (D) SKOV-3 cells were pretreated for 1 h with increasing concentrations of the metalloproteinase inhibitor GM6001 before they were harvested and subjected to Western blot. The data shown are representative of at least three independent experiments. Data were analyzed by Student’s t -test. * p
    Figure Legend Snippet: Effect of Src and MMP inhibitors on EGFR phosphorylation. (A) SKOV-3 cells were treated with 100 nM PAF for 0, 5, 10, 15, 20, or 25 min. Following PAF treatment, cells were lysed and lysates were evaluated by Western blotting. Data were normalized to total Src protein expression and are expressed as the fold change (average ± S.E.M.) in phospho-Src compared to vehicle-treated cells. Representative blots for phosphor/total-EGFR are shown. (B) SKOV-3 cells were treated with the Src inhibitor PP2 (20 μM) for 1 hour before stimulation with PAF (100 nM) or EGF (5 ng/ml) for 5 min. Cells were harvested and subjected to Western blot. (C) SKOV-3 cells were pretreated for 1 h with the metalloproteinase inhibitor GM6001 (20 μM). Cells were then stimulated with either PAF (100 nM) or EGF (5 ng/ml) for 5 min before they were harvested and immunoblotting. (D) SKOV-3 cells were pretreated for 1 h with increasing concentrations of the metalloproteinase inhibitor GM6001 before they were harvested and subjected to Western blot. The data shown are representative of at least three independent experiments. Data were analyzed by Student’s t -test. * p

    Techniques Used: Western Blot, Expressing

    17) Product Images from "Thrombin induces heme oxygenase-1 expression in human synovial fibroblasts through protease-activated receptor signaling pathways"

    Article Title: Thrombin induces heme oxygenase-1 expression in human synovial fibroblasts through protease-activated receptor signaling pathways

    Journal: Arthritis Research & Therapy

    doi: 10.1186/ar3815

    Thrombin induced Nrf2 activation in synovial fibroblasts . (A) Osteoarthritis synovial fibroblasts (OASFs; six OA lines) were transfected with Nrf2 siRNA for 24 hours, and HO-1 expression was examined with qPCR. (B) OASFs (six OA lines) were incubated with thrombin for indicated time intervals, and Nrf2 phosphorylation was examined with Western blotting. (C) Cells (five OA lines) were pretreated 30 minutes with PPACK, rottlerin, or PP2 followed by stimulation with thrombin for 30 minutes, and Nrf2 phosphorylation was examined with Western blotting. Results are expressed as the mean ± SEM. * P
    Figure Legend Snippet: Thrombin induced Nrf2 activation in synovial fibroblasts . (A) Osteoarthritis synovial fibroblasts (OASFs; six OA lines) were transfected with Nrf2 siRNA for 24 hours, and HO-1 expression was examined with qPCR. (B) OASFs (six OA lines) were incubated with thrombin for indicated time intervals, and Nrf2 phosphorylation was examined with Western blotting. (C) Cells (five OA lines) were pretreated 30 minutes with PPACK, rottlerin, or PP2 followed by stimulation with thrombin for 30 minutes, and Nrf2 phosphorylation was examined with Western blotting. Results are expressed as the mean ± SEM. * P

    Techniques Used: Activation Assay, Transfection, Expressing, Real-time Polymerase Chain Reaction, Incubation, Western Blot

    c-Src is involved in thrombin-induced HO-1 expression in synovial fibroblasts . (A, B) Osteoarthritis synovial fibroblasts (OASFs) (six OA lines) were pretreated for 30 minutes with PP2 (3 μ M ) or transfected with c-Src mutant for 24 hours followed by stimulation with thrombin for 24 hours, and the HO-1 expression was examined with qPCR and Western blotting. (C) OASFs (five OA lines) were incubated with thrombin for indicated time intervals, and c-Src phosphorylation was examined with Western blotting. (D) Cells (six OA lines) were pretreated 30 minutes with PPACK or rottlerin, followed by stimulation with thrombin for 30 minutes, and c-Src phosphorylation was examined with Western blotting. Results are expressed as the mean ± SEM. * P
    Figure Legend Snippet: c-Src is involved in thrombin-induced HO-1 expression in synovial fibroblasts . (A, B) Osteoarthritis synovial fibroblasts (OASFs) (six OA lines) were pretreated for 30 minutes with PP2 (3 μ M ) or transfected with c-Src mutant for 24 hours followed by stimulation with thrombin for 24 hours, and the HO-1 expression was examined with qPCR and Western blotting. (C) OASFs (five OA lines) were incubated with thrombin for indicated time intervals, and c-Src phosphorylation was examined with Western blotting. (D) Cells (six OA lines) were pretreated 30 minutes with PPACK or rottlerin, followed by stimulation with thrombin for 30 minutes, and c-Src phosphorylation was examined with Western blotting. Results are expressed as the mean ± SEM. * P

    Techniques Used: Expressing, Transfection, Mutagenesis, Real-time Polymerase Chain Reaction, Western Blot, Incubation

    PAR, PKCδ, and c-Src are involved in thrombin-induced Nrf2 activation . (A) Osteoarthritis synovial fibroblast (OASFs; seven OA lines) were pretreated with GF109203X, rottlerin, and PP2 for 30 minutes followed by stimulation with thrombin for 120 minutes, and ChIP assay was then performed. Chromatin was immunoprecipitated with anti-Nrf2 antibody. One percent of the precipitated chromatin was assayed to verify equal loading (Input). OASFs (eight OA lines) were incubated with thrombin for 24 hours (B) or pretreated with PPACK, GF109203X, rottlerin, and PP2 for 30 minutes (C) or co-transfected with PAR1 siRNA, PAR3 siRNA, PAR4 siRNA, PKCδ siRNA, and c-Src mutant for 24 hours (D) before incubation with thrombin for 24 hours. HO-1 luciferase activity was then assayed. Results are expressed as the mean ± SEM. * P
    Figure Legend Snippet: PAR, PKCδ, and c-Src are involved in thrombin-induced Nrf2 activation . (A) Osteoarthritis synovial fibroblast (OASFs; seven OA lines) were pretreated with GF109203X, rottlerin, and PP2 for 30 minutes followed by stimulation with thrombin for 120 minutes, and ChIP assay was then performed. Chromatin was immunoprecipitated with anti-Nrf2 antibody. One percent of the precipitated chromatin was assayed to verify equal loading (Input). OASFs (eight OA lines) were incubated with thrombin for 24 hours (B) or pretreated with PPACK, GF109203X, rottlerin, and PP2 for 30 minutes (C) or co-transfected with PAR1 siRNA, PAR3 siRNA, PAR4 siRNA, PKCδ siRNA, and c-Src mutant for 24 hours (D) before incubation with thrombin for 24 hours. HO-1 luciferase activity was then assayed. Results are expressed as the mean ± SEM. * P

    Techniques Used: Activation Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Incubation, Transfection, Mutagenesis, Luciferase, Activity Assay

    18) Product Images from "Non-Canonical Activation of the Epidermal Growth Factor Receptor by Carbon Nanoparticles"

    Article Title: Non-Canonical Activation of the Epidermal Growth Factor Receptor by Carbon Nanoparticles

    Journal: Nanomaterials

    doi: 10.3390/nano8040267

    Caveolin-1 and EGFR co-localization as a feature of non-canonical EGFR activation. Epithelial cells (RLE-6TN) were exposed (5 min) to carbon nanoparticles (CNP), non-nano carbon particles (CP), each 10 µg/cm 2 , (50 µM) H 2 O 2 , (5 µM) C6 ceramide, or EGF (100 ng/mL), respectively. ( A ) Subcellular localization of EGFR (red Alexa flour 594) and caveolin-1 (green Alexa flour 488). Co-localization is visualized by the yellow color in merged images; ( B ) Subcellular localization of EGFR and caveolin-1 in cells pre-treated with inhibitors of carbon nanoparticle-specific signaling prior to particle or EGF exposure: SFK inhibitor PP2 (10 µM), 1 mM ectoine ( E ), 75 µM α-tocopherol (Toco), and 1 µg/mL filipin III (Fil). Co-localization is visualized by the yellow color in merged images; ( C ) Subcellular localization of caveolin-1 (red Alexa fluor 594) after exposure to carbon nanoparticles (CNP), carbon particles (CP), or hydrogen peroxide (H 2 O 2 ); ( D ) Quantification and representative Western blots of caveolin-1 in lipid raft fraction of RLE-6TN cells exposed to CNP (10 µg/cm 2 ). Raft and non-raft fraction were isolated from density gradients after ultracentrifugation. Pre-treatment of cells with α-tocopherol (Toco) was applied as an antioxidant strategy. GAPDH was used as a control protein not associated with lipid rafts. The bars in the graph represent the additive immune signals of raft and non-raft fractions, which was indicated in the representative original Western blots; ( E ) Quantification and representative Western-blot of EGFR phosphorylation at Tyr 845 . Nuclei were stained with DAPI (blue). Scale bars represent 20 µm; *, which was significantly different to PBS control ( p
    Figure Legend Snippet: Caveolin-1 and EGFR co-localization as a feature of non-canonical EGFR activation. Epithelial cells (RLE-6TN) were exposed (5 min) to carbon nanoparticles (CNP), non-nano carbon particles (CP), each 10 µg/cm 2 , (50 µM) H 2 O 2 , (5 µM) C6 ceramide, or EGF (100 ng/mL), respectively. ( A ) Subcellular localization of EGFR (red Alexa flour 594) and caveolin-1 (green Alexa flour 488). Co-localization is visualized by the yellow color in merged images; ( B ) Subcellular localization of EGFR and caveolin-1 in cells pre-treated with inhibitors of carbon nanoparticle-specific signaling prior to particle or EGF exposure: SFK inhibitor PP2 (10 µM), 1 mM ectoine ( E ), 75 µM α-tocopherol (Toco), and 1 µg/mL filipin III (Fil). Co-localization is visualized by the yellow color in merged images; ( C ) Subcellular localization of caveolin-1 (red Alexa fluor 594) after exposure to carbon nanoparticles (CNP), carbon particles (CP), or hydrogen peroxide (H 2 O 2 ); ( D ) Quantification and representative Western blots of caveolin-1 in lipid raft fraction of RLE-6TN cells exposed to CNP (10 µg/cm 2 ). Raft and non-raft fraction were isolated from density gradients after ultracentrifugation. Pre-treatment of cells with α-tocopherol (Toco) was applied as an antioxidant strategy. GAPDH was used as a control protein not associated with lipid rafts. The bars in the graph represent the additive immune signals of raft and non-raft fractions, which was indicated in the representative original Western blots; ( E ) Quantification and representative Western-blot of EGFR phosphorylation at Tyr 845 . Nuclei were stained with DAPI (blue). Scale bars represent 20 µm; *, which was significantly different to PBS control ( p

    Techniques Used: Activation Assay, Western Blot, Isolation, Staining

    19) Product Images from "Claudin-1 expression confers resistance to anoikis in colon cancer cells in a Src-dependent manner"

    Article Title: Claudin-1 expression confers resistance to anoikis in colon cancer cells in a Src-dependent manner

    Journal: Carcinogenesis

    doi: 10.1093/carcin/bgs275

    Effect of the modulation of Src activation upon claudin-1-dependent resistance to anoikis. (A) Effect of PP2 treatment (10 or 20 µM) in SW480 claudin-1 cells upon Src phosphorylation and anoikis using the cell-death enzyme-linked immunosorbent
    Figure Legend Snippet: Effect of the modulation of Src activation upon claudin-1-dependent resistance to anoikis. (A) Effect of PP2 treatment (10 or 20 µM) in SW480 claudin-1 cells upon Src phosphorylation and anoikis using the cell-death enzyme-linked immunosorbent

    Techniques Used: Activation Assay

    20) Product Images from "An inhibitory role for FAK in regulating proliferation: a link between limited adhesion and RhoA-ROCK signaling"

    Article Title: An inhibitory role for FAK in regulating proliferation: a link between limited adhesion and RhoA-ROCK signaling

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200510062

    FRNK stimulates proliferation in low adhesive contexts. (A) Western blots of GFP-, FAK-, FRNK-, or FAK-Y397F-overexpressing ECs in spread (substrates coated with 25 μg/ml fibronectin) or unspread (substrates patterned with 625-μm 2 islands of fibronectin) conditions and probed for phospho–Y397-FAK, total FAK, or GAPDH. (B) Western blot of phospho–Y397-FAK and total FAK in the Triton X-100–insoluble fraction of unspread ECs expressing GFP (control), FRNK, FAK, or FAK-Y397F. β–actin is shown as a loading control. (C and D) Graph of the percentage of GFP-, FAK-, FRNK-, FAT-, or FAK-Y397F–overexpressing ECs entering S phase when cultured on 625-μm 2 islands of fibronectin (C) or on substrates coated with 25 μg/ml fibronectin (D). (E) Graph of the percentage of FAK−/− cells, FAK-reexpressing cells, and FAK−/− cells overexpressing FAK-Y397F in S phase when cultured in spread or unspread conditions. (F) Graph of the percentage of GFP-, FAK-, or FRNK-overexpressing ECs entering S phase when cultured on 625-μm 2 islands of fibronectin and treated with either 10 μM UO126 or 1 μM PP2. All data is expressed as ± SEM for three independent experiments. *, P
    Figure Legend Snippet: FRNK stimulates proliferation in low adhesive contexts. (A) Western blots of GFP-, FAK-, FRNK-, or FAK-Y397F-overexpressing ECs in spread (substrates coated with 25 μg/ml fibronectin) or unspread (substrates patterned with 625-μm 2 islands of fibronectin) conditions and probed for phospho–Y397-FAK, total FAK, or GAPDH. (B) Western blot of phospho–Y397-FAK and total FAK in the Triton X-100–insoluble fraction of unspread ECs expressing GFP (control), FRNK, FAK, or FAK-Y397F. β–actin is shown as a loading control. (C and D) Graph of the percentage of GFP-, FAK-, FRNK-, FAT-, or FAK-Y397F–overexpressing ECs entering S phase when cultured on 625-μm 2 islands of fibronectin (C) or on substrates coated with 25 μg/ml fibronectin (D). (E) Graph of the percentage of FAK−/− cells, FAK-reexpressing cells, and FAK−/− cells overexpressing FAK-Y397F in S phase when cultured in spread or unspread conditions. (F) Graph of the percentage of GFP-, FAK-, or FRNK-overexpressing ECs entering S phase when cultured on 625-μm 2 islands of fibronectin and treated with either 10 μM UO126 or 1 μM PP2. All data is expressed as ± SEM for three independent experiments. *, P

    Techniques Used: Western Blot, Expressing, Cell Culture

    21) Product Images from "SHP-2 activates signaling of the nuclear factor of activated T cells to promote skeletal muscle growth"

    Article Title: SHP-2 activates signaling of the nuclear factor of activated T cells to promote skeletal muscle growth

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200602029

    SHP-2 mediates myotube multinucleation in an SFK- and integrin-dependent manner. (A) After 24 h in differentiation medium (DM), cells were treated for 24 and 48 h either with 2 μM PP2 (+PP2) or without PP2 (−PP2). Lysates were subjected to immunoprecipitation with anti–SIRP-1α or anti–SHP-2 antibodies. Immune complexes were immunoblotted with antiphosphotyrosine, anti–SIRP-1α, and anti–SHP-2 antibodies. (B) Representative photomicrographs of PP2-treated C2C12 myoblasts as described in A. The number of myotubes containing more than two nuclei was calculated, and the results represent the mean percentages ± SEM (error bars) of three independent experiments (*, P
    Figure Legend Snippet: SHP-2 mediates myotube multinucleation in an SFK- and integrin-dependent manner. (A) After 24 h in differentiation medium (DM), cells were treated for 24 and 48 h either with 2 μM PP2 (+PP2) or without PP2 (−PP2). Lysates were subjected to immunoprecipitation with anti–SIRP-1α or anti–SHP-2 antibodies. Immune complexes were immunoblotted with antiphosphotyrosine, anti–SIRP-1α, and anti–SHP-2 antibodies. (B) Representative photomicrographs of PP2-treated C2C12 myoblasts as described in A. The number of myotubes containing more than two nuclei was calculated, and the results represent the mean percentages ± SEM (error bars) of three independent experiments (*, P

    Techniques Used: Immunoprecipitation

    22) Product Images from "Preconditioning-mimetics Bradykinin and DADLE Activate PI3-kinase Through Divergent Pathways"

    Article Title: Preconditioning-mimetics Bradykinin and DADLE Activate PI3-kinase Through Divergent Pathways

    Journal: Journal of molecular and cellular cardiology

    doi: 10.1016/j.yjmcc.2007.01.004

    Infarct size as a percentage of the risk zone following a 30-min coronary occlusion/2-h reperfusion in isolated rabbit hearts. Open circles represent individual data points, while closed circles represent group averages with SEM. Bradykinin salvaged ischemic myocardium. Metalloproteinase inhibitor III (MPI) had no effect on bradykinin’s cardioprotective effect suggesting cleavage of heparin binding epidermal growth factor-like growth factor and therefore subsequent transactivation of the epidermal growth factor receptor were not essential for bradykinin’s protective effect. These results distinguish bradykinin from DADLE. However, both PP2 and wortmannin, Src kinase and phosphatidylinositol 3-kinase antagonists, resp., did block bradykinin’s salutary effect implicating these kinases in the downstream signaling cascade. The latter observations suggest bradykinin and DADLE do have signaling steps in common.
    Figure Legend Snippet: Infarct size as a percentage of the risk zone following a 30-min coronary occlusion/2-h reperfusion in isolated rabbit hearts. Open circles represent individual data points, while closed circles represent group averages with SEM. Bradykinin salvaged ischemic myocardium. Metalloproteinase inhibitor III (MPI) had no effect on bradykinin’s cardioprotective effect suggesting cleavage of heparin binding epidermal growth factor-like growth factor and therefore subsequent transactivation of the epidermal growth factor receptor were not essential for bradykinin’s protective effect. These results distinguish bradykinin from DADLE. However, both PP2 and wortmannin, Src kinase and phosphatidylinositol 3-kinase antagonists, resp., did block bradykinin’s salutary effect implicating these kinases in the downstream signaling cascade. The latter observations suggest bradykinin and DADLE do have signaling steps in common.

    Techniques Used: Isolation, Binding Assay, Blocking Assay

    DADLE significantly increased production of reactive oxygen species by adult rabbit ventricular myocytes. This increased production was blocked when cells were additionally exposed to either PP2, an antagonist of Src kinase, or wortmannin, an inhibitor of phosphatidylinositol 3-kinase. Neither PP2 nor wortmannin alone had any influence.
    Figure Legend Snippet: DADLE significantly increased production of reactive oxygen species by adult rabbit ventricular myocytes. This increased production was blocked when cells were additionally exposed to either PP2, an antagonist of Src kinase, or wortmannin, an inhibitor of phosphatidylinositol 3-kinase. Neither PP2 nor wortmannin alone had any influence.

    Techniques Used:

    Effect of DADLE, metalloproteinase inhibitor III (MPI), and PP2 on infarct size expressed as a percentage of the risk zone in isolated rabbit hearts. Open circles represent individual data points, while closed circles represent group averages with SEM. DADLE significantly decreased infarction following a 30-min coronary occlusion/2-h reperfusion, and this salutary effect was aborted by both MPI and the Src kinase antagonist PP2. These results support involvement of transactivation of the epidermal growth factor receptor in the intracellular signaling triggered by DADLE.
    Figure Legend Snippet: Effect of DADLE, metalloproteinase inhibitor III (MPI), and PP2 on infarct size expressed as a percentage of the risk zone in isolated rabbit hearts. Open circles represent individual data points, while closed circles represent group averages with SEM. DADLE significantly decreased infarction following a 30-min coronary occlusion/2-h reperfusion, and this salutary effect was aborted by both MPI and the Src kinase antagonist PP2. These results support involvement of transactivation of the epidermal growth factor receptor in the intracellular signaling triggered by DADLE.

    Techniques Used: Isolation

    23) Product Images from "Endocytosis of somatodendritic NCKX2 is regulated by Src family kinase-dependent tyrosine phosphorylation"

    Article Title: Endocytosis of somatodendritic NCKX2 is regulated by Src family kinase-dependent tyrosine phosphorylation

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2013.00014

    Src family kinase (SFK)-mediated Tyr-365 phosphorylation of NCKX2 regulates its surface expression. (A) Tyrosine phosphorylation of NCKX2 in response to carbachol (CCh) in PC-12 cells. We transfected FLAG-tagged NCKX2-WT or NCKX2-Y365A into PC-12 cells which normally express PYK2 and SFK, and treated the cells with 1 mM CCh for 2 min. To observe tyrosine-phosphorylation of NCKX2, NCKX2 was immunoprecipitated with anti-FLAG and then immunoblotted with anti-phosphotyrosine (pTyr) IgG. NCKX2-WT, but not NCKX2-Y365A, was strongly phosphorylated by CCh treatment. Pretreatment with 10 μM PP2, a selective inhibitor of SFK, reduced the CCh-induced tyrosine phosphorylation of NCKX2-WT. The FLAG signals on the same blot are shown in the lower image, showing little difference in expression levels of wild-type or Y365A mutant NCKX2 between different conditions. (B) Hippocampal neurons expressing FLAG-tagged NCKX2-WT (Ba) or NCKX2-Y365A (Bb) were treated with 0.1% DMSO (control; upper) or 10 μM PP2 (bottom) for 2 h. Wild-type or Y365A mutant of NCKX2 expressed on the surface was visualized with anti-FLAG (s-FLAG; green). Dendrites were immunostained with anti-MAP2 (blue). Arrowheads indicate axons. Scale bar: 50 μm. (C) The spatially averaged fluorescence intensity of wild-type (Ca) or Y365A mutant (Cb) of surface NCKX2 on SD- or axonal ROIs of the vehicle (DMSO; black)- or PP2 (gray)-treated neurons under the same imaging settings. ** p
    Figure Legend Snippet: Src family kinase (SFK)-mediated Tyr-365 phosphorylation of NCKX2 regulates its surface expression. (A) Tyrosine phosphorylation of NCKX2 in response to carbachol (CCh) in PC-12 cells. We transfected FLAG-tagged NCKX2-WT or NCKX2-Y365A into PC-12 cells which normally express PYK2 and SFK, and treated the cells with 1 mM CCh for 2 min. To observe tyrosine-phosphorylation of NCKX2, NCKX2 was immunoprecipitated with anti-FLAG and then immunoblotted with anti-phosphotyrosine (pTyr) IgG. NCKX2-WT, but not NCKX2-Y365A, was strongly phosphorylated by CCh treatment. Pretreatment with 10 μM PP2, a selective inhibitor of SFK, reduced the CCh-induced tyrosine phosphorylation of NCKX2-WT. The FLAG signals on the same blot are shown in the lower image, showing little difference in expression levels of wild-type or Y365A mutant NCKX2 between different conditions. (B) Hippocampal neurons expressing FLAG-tagged NCKX2-WT (Ba) or NCKX2-Y365A (Bb) were treated with 0.1% DMSO (control; upper) or 10 μM PP2 (bottom) for 2 h. Wild-type or Y365A mutant of NCKX2 expressed on the surface was visualized with anti-FLAG (s-FLAG; green). Dendrites were immunostained with anti-MAP2 (blue). Arrowheads indicate axons. Scale bar: 50 μm. (C) The spatially averaged fluorescence intensity of wild-type (Ca) or Y365A mutant (Cb) of surface NCKX2 on SD- or axonal ROIs of the vehicle (DMSO; black)- or PP2 (gray)-treated neurons under the same imaging settings. ** p

    Techniques Used: Expressing, Transfection, Immunoprecipitation, Mutagenesis, Fluorescence, Imaging

    24) Product Images from "Src Kinase Dependent Rapid Non-genomic Modulation of Hippocampal Spinogenesis Induced by Androgen and Estrogen"

    Article Title: Src Kinase Dependent Rapid Non-genomic Modulation of Hippocampal Spinogenesis Induced by Androgen and Estrogen

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2018.00282

    Effects of Src kinase blocker (PP2) on DHT-induced spine increase and change in morphology in hippocampal slices. (A) Spines were analyzed along the secondary dendrites of pyramidal neurons in the stratum radiatum of CA1 neurons. Dendrite after DHT-treatment for 2 h (DHT) and dendrite after DHT plus PP2 treatment for 2 h (PP2+DHT). (Spiso) shows the image of dendrite and spines analyzed with Spiso-3D software. Maximal intensity projections onto XY plane is shown. Traced dendrite is shown in red color and spines are indicated in yellow color. (Model) shows 3 dimensional model illustration of (Spiso) image. Bar, 5 μm. (B) Effect of treatments by DHT or PP2 on the total spine density in CA1 neurons. Vertical axis is the average number of spines per 1 μm of dendrite. A 2 h treatment in ACSF without drugs (Control), with 10 nM DHT (DHT), with 10 nM DHT and 10 μM PP2 (PP2 + DHT), and with PP2 only (PP2). (C) Histogram of spine head diameters after a 2 h treatment in ACSF without drugs (Control, black dashed line), with 10 nM DHT (black line), with 10 nM DHT and 10 μM PP2 (red line). Spines were classified into three categories depending on their head diameter, e.g., 0.2–0.4 μm as small-head spines, 0.4–0.5 μm as middle-head spines, and larger than 0.5 μm as large-head spines. Vertical axis is the number of spines per 1 μm of dendrite. (D) Density of three subtypes of spines. Abbreviations are same as in (B) . Vertical axis is the number of spines per 1 μm of dendrite. From left to right, small-head spines (small), middle-head spines (middle), and large-head spines (large) type. ACSF without drugs (Control, open column), DHT (black column), PP2 + DHT (red column). Vertical axis is the number of spines per 1 μm of dendrite. Results are represented as mean ± SEM. Statistical significance yielded * P
    Figure Legend Snippet: Effects of Src kinase blocker (PP2) on DHT-induced spine increase and change in morphology in hippocampal slices. (A) Spines were analyzed along the secondary dendrites of pyramidal neurons in the stratum radiatum of CA1 neurons. Dendrite after DHT-treatment for 2 h (DHT) and dendrite after DHT plus PP2 treatment for 2 h (PP2+DHT). (Spiso) shows the image of dendrite and spines analyzed with Spiso-3D software. Maximal intensity projections onto XY plane is shown. Traced dendrite is shown in red color and spines are indicated in yellow color. (Model) shows 3 dimensional model illustration of (Spiso) image. Bar, 5 μm. (B) Effect of treatments by DHT or PP2 on the total spine density in CA1 neurons. Vertical axis is the average number of spines per 1 μm of dendrite. A 2 h treatment in ACSF without drugs (Control), with 10 nM DHT (DHT), with 10 nM DHT and 10 μM PP2 (PP2 + DHT), and with PP2 only (PP2). (C) Histogram of spine head diameters after a 2 h treatment in ACSF without drugs (Control, black dashed line), with 10 nM DHT (black line), with 10 nM DHT and 10 μM PP2 (red line). Spines were classified into three categories depending on their head diameter, e.g., 0.2–0.4 μm as small-head spines, 0.4–0.5 μm as middle-head spines, and larger than 0.5 μm as large-head spines. Vertical axis is the number of spines per 1 μm of dendrite. (D) Density of three subtypes of spines. Abbreviations are same as in (B) . Vertical axis is the number of spines per 1 μm of dendrite. From left to right, small-head spines (small), middle-head spines (middle), and large-head spines (large) type. ACSF without drugs (Control, open column), DHT (black column), PP2 + DHT (red column). Vertical axis is the number of spines per 1 μm of dendrite. Results are represented as mean ± SEM. Statistical significance yielded * P

    Techniques Used: Software

    25) Product Images from "Src regulates amino acid-mediated mTORC1 activation by disrupting GATOR1-Rag GTPase interaction"

    Article Title: Src regulates amino acid-mediated mTORC1 activation by disrupting GATOR1-Rag GTPase interaction

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06844-4

    Src regulates cell growth, TFEB localization and autophagy. a , b The histogram shows the cell size of SH-SY5Y ( a ) and MEF ( b ) cells treated with DMSO or PP2 (5 μM for 24 h). The bar diagram represents the mean diameter of at least 10 4 cells. Error bars represent values in ± SEM. *** p
    Figure Legend Snippet: Src regulates cell growth, TFEB localization and autophagy. a , b The histogram shows the cell size of SH-SY5Y ( a ) and MEF ( b ) cells treated with DMSO or PP2 (5 μM for 24 h). The bar diagram represents the mean diameter of at least 10 4 cells. Error bars represent values in ± SEM. *** p

    Techniques Used:

    Src regulates mTORC1 activity independently of TSC2. a WT ( Tsc2 +/+ ) and TSC2 null (Tsc2 −/− ) MEF cells were coimmunostained for DAPI (blue) and TSC2 (green). Bar indicates 40 μm. b Amino acid-starved Tsc2 −/− MEFs showed significantly higher activity of mTORC1 compared to Tsc2 +/+ MEFs. Activation was further potentiated upon stimulation with amino acids. Lysates were probed with antibodies as indicated. c Tsc2 +/+ and Tsc2 −/− MEFs cells were starved and treated with vehicle (DMSO) or PP2 (10 μM) for the last 2 h of starvation and then stimulated with amino acids (30 min). Lysates were probed with antibodies as indicated. d . WCE stands for whole-cell extract. Quantified data represent means ± SEM of n = 2–3 independent experiments. e , f MEF cells were transfected to express Src, starved and stimulated with amino acids (30 min) prior to immunofluorescent labeling of RagC (red) and Src (green) in e and LAMP1 (green) and Src (red) in f . Representative cells are shown where yellow or orange pixels indicate co-localization in the merged images. In all images, insets show selected fields that were magnified by a factor of 4. Bar indicates 40 μm. g HEK cells, transiently transfected with HA-GST-RagA and HA-GST-RagC, were lysed and co-IP analyses were performed to test interaction of Src with the Rags. Immunoblot analyses were used to measure the levels of the indicated proteins. h HEK cells were lysed and coIP analyses were performed to test interaction of Src with the Rags. i HEK cells were starved prior to amino acid stimulation (30 min). Cells were lysed and coIP analyses were performed. IgG antibody pulldown was performed as a negative control in g to i . GAPDH and tubulin were used as a loading control in immunoblot assays
    Figure Legend Snippet: Src regulates mTORC1 activity independently of TSC2. a WT ( Tsc2 +/+ ) and TSC2 null (Tsc2 −/− ) MEF cells were coimmunostained for DAPI (blue) and TSC2 (green). Bar indicates 40 μm. b Amino acid-starved Tsc2 −/− MEFs showed significantly higher activity of mTORC1 compared to Tsc2 +/+ MEFs. Activation was further potentiated upon stimulation with amino acids. Lysates were probed with antibodies as indicated. c Tsc2 +/+ and Tsc2 −/− MEFs cells were starved and treated with vehicle (DMSO) or PP2 (10 μM) for the last 2 h of starvation and then stimulated with amino acids (30 min). Lysates were probed with antibodies as indicated. d . WCE stands for whole-cell extract. Quantified data represent means ± SEM of n = 2–3 independent experiments. e , f MEF cells were transfected to express Src, starved and stimulated with amino acids (30 min) prior to immunofluorescent labeling of RagC (red) and Src (green) in e and LAMP1 (green) and Src (red) in f . Representative cells are shown where yellow or orange pixels indicate co-localization in the merged images. In all images, insets show selected fields that were magnified by a factor of 4. Bar indicates 40 μm. g HEK cells, transiently transfected with HA-GST-RagA and HA-GST-RagC, were lysed and co-IP analyses were performed to test interaction of Src with the Rags. Immunoblot analyses were used to measure the levels of the indicated proteins. h HEK cells were lysed and coIP analyses were performed to test interaction of Src with the Rags. i HEK cells were starved prior to amino acid stimulation (30 min). Cells were lysed and coIP analyses were performed. IgG antibody pulldown was performed as a negative control in g to i . GAPDH and tubulin were used as a loading control in immunoblot assays

    Techniques Used: Activity Assay, Activation Assay, Transfection, Labeling, Co-Immunoprecipitation Assay, Negative Control

    Src kinase disrupts the interaction of GATOR1 with the Rags. a Src kinase interacts with the GATOR1 complex. HEK293 cells, transiently transfected with GFP-Depdc5, were lysed and subjected to immunoprecipation with antibodies against Src or IgG (negative control) followed by immunoblotting for the indicated proteins. Lysates were probed with antibodies as indicated. b HEK cells, transiently transfected with scramble shRNA or shRNAs targeting DEPDC5 , NPRL2 , and NPRL3 genes for 48 h prior to starvation and treatment with vehicle (DMSO) or PP2 for the last 2 h of starvation and then stimulated with amino acids (30 min). Immunoblot analyses were performed to measure the levels of the indicated proteins and phosphorylation states. c HEK293 cells, transiently transfected with the components of GATOR1 and constitutively active Src (CA-Src), were lysed and coIP analyses were performed to test interaction of GATOR1 with RagA, RagB, or RagC. d HEK293 cells, transiently transfected with GATOR1 components, CA-Src or kinase-dead Src (K295R), were lysed and coIP analyses were performed to test interaction of GATOR1 with RagA or RagC. e HEK293 cells, transiently transfected with the components of GATOR1 and wild-type Src (WT-Src), were lysed and subjected to RagC immunoprecipitation (IP) followed by immunoblotting for the indicated proteins. Lysates were probed with antibodies as indicated. f Inhibition of Src kinase promotes GATOR1–Rag interaction. HEK293 cells transfected as in e were treated with PP2 (5 μM, 24 h) prior to lysis, and then subjected to RagC immunoprecipitation (IP) followed by immunoblotting for the indicated proteins. GAPDH was used as a loading control in all immunoblot assays
    Figure Legend Snippet: Src kinase disrupts the interaction of GATOR1 with the Rags. a Src kinase interacts with the GATOR1 complex. HEK293 cells, transiently transfected with GFP-Depdc5, were lysed and subjected to immunoprecipation with antibodies against Src or IgG (negative control) followed by immunoblotting for the indicated proteins. Lysates were probed with antibodies as indicated. b HEK cells, transiently transfected with scramble shRNA or shRNAs targeting DEPDC5 , NPRL2 , and NPRL3 genes for 48 h prior to starvation and treatment with vehicle (DMSO) or PP2 for the last 2 h of starvation and then stimulated with amino acids (30 min). Immunoblot analyses were performed to measure the levels of the indicated proteins and phosphorylation states. c HEK293 cells, transiently transfected with the components of GATOR1 and constitutively active Src (CA-Src), were lysed and coIP analyses were performed to test interaction of GATOR1 with RagA, RagB, or RagC. d HEK293 cells, transiently transfected with GATOR1 components, CA-Src or kinase-dead Src (K295R), were lysed and coIP analyses were performed to test interaction of GATOR1 with RagA or RagC. e HEK293 cells, transiently transfected with the components of GATOR1 and wild-type Src (WT-Src), were lysed and subjected to RagC immunoprecipitation (IP) followed by immunoblotting for the indicated proteins. Lysates were probed with antibodies as indicated. f Inhibition of Src kinase promotes GATOR1–Rag interaction. HEK293 cells transfected as in e were treated with PP2 (5 μM, 24 h) prior to lysis, and then subjected to RagC immunoprecipitation (IP) followed by immunoblotting for the indicated proteins. GAPDH was used as a loading control in all immunoblot assays

    Techniques Used: Transfection, Negative Control, shRNA, Co-Immunoprecipitation Assay, Immunoprecipitation, Inhibition, Lysis

    Src promotes mTORC1 recruitment to lysosomes. a HEK cells transiently transfected with scrambled shRNA or sh Src prior to coIP analyses. Lysates were used for immunoprecipitation by either Src or IgG antibody (negative control). b , c HEK293 cells were starved and treated with vehicle (DMSO) or PP2 (10 μM) for the last 2 h of starvation and then stimulated with amino acids (30 min). coIP analyses were performed to test interaction of mTOR and Raptor with RagA in b or RagB in c . Immunoblot analyses were used to measure the levels of the indicated proteins and phosphorylation states. d HEK293 cells stably expressing Flag-raptor, transiently transfected with HA-GST-RagA WT /C WT or HA-GST-RagA GTP /C GDP , were starved and treated with vehicle (DMSO) or PP2 (10 μM) for the last 2 h of starvation and then stimulated with amino acids (30 min). coIP analyses were performed to test interaction of mTOR and Raptor with RagA/C. Immunoblot analyses were used to measure the levels of the indicated proteins. e , f HEK293 cells were transiently transfected with HA-GST-RagA WT /C WT ( e ) or HA-GST-RagA GTP /C GDP ( f ) and treated as in a prior to immunofluorescence labeling of HA (green) and endogenous mTOR (red). Representative cells are shown where punctate structures indicate lysosomal localization of mTOR. Magnified images are represented in white boxes. Bar indicates 40 μm. g HEK293 cells stably expressing HA-GST-RagA WT /C WT or HA-GST-RagA GTP /C GDP were treated as in b . Immunoblot analyses were used to measure the levels of the indicated proteins and phosphorylation states. Short and long indicate short and long exposure, respectively. Box plots represent SE of n = 3 independent experiments. *** p
    Figure Legend Snippet: Src promotes mTORC1 recruitment to lysosomes. a HEK cells transiently transfected with scrambled shRNA or sh Src prior to coIP analyses. Lysates were used for immunoprecipitation by either Src or IgG antibody (negative control). b , c HEK293 cells were starved and treated with vehicle (DMSO) or PP2 (10 μM) for the last 2 h of starvation and then stimulated with amino acids (30 min). coIP analyses were performed to test interaction of mTOR and Raptor with RagA in b or RagB in c . Immunoblot analyses were used to measure the levels of the indicated proteins and phosphorylation states. d HEK293 cells stably expressing Flag-raptor, transiently transfected with HA-GST-RagA WT /C WT or HA-GST-RagA GTP /C GDP , were starved and treated with vehicle (DMSO) or PP2 (10 μM) for the last 2 h of starvation and then stimulated with amino acids (30 min). coIP analyses were performed to test interaction of mTOR and Raptor with RagA/C. Immunoblot analyses were used to measure the levels of the indicated proteins. e , f HEK293 cells were transiently transfected with HA-GST-RagA WT /C WT ( e ) or HA-GST-RagA GTP /C GDP ( f ) and treated as in a prior to immunofluorescence labeling of HA (green) and endogenous mTOR (red). Representative cells are shown where punctate structures indicate lysosomal localization of mTOR. Magnified images are represented in white boxes. Bar indicates 40 μm. g HEK293 cells stably expressing HA-GST-RagA WT /C WT or HA-GST-RagA GTP /C GDP were treated as in b . Immunoblot analyses were used to measure the levels of the indicated proteins and phosphorylation states. Short and long indicate short and long exposure, respectively. Box plots represent SE of n = 3 independent experiments. *** p

    Techniques Used: Transfection, shRNA, Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control, Stable Transfection, Expressing, Immunofluorescence, Labeling

    26) Product Images from "Early integrin binding to Arg-Gly-Asp peptide activates actin polymerization and contractile movement that stimulates outward translocation"

    Article Title: Early integrin binding to Arg-Gly-Asp peptide activates actin polymerization and contractile movement that stimulates outward translocation

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1109485108

    Outward movement of integrin clusters follows protrusion of cell edges. ( A ) Long-ranged outward then inward translocation of ligated integrin complexes. ( B ) Preincubation of 10 μM PP2, a Src-kinase inhibitor, blocked outward movement and cell spreading. Inset overlay: bright-field images, respectively. Kymographs: lateral movement of marked clusters, respectively. ( C , D ) Radial positions of each cluster’s trajectory (colored aster) and averaged cell edges (yellow triangle) of (A, 27 clusters) and (B, 8 clusters). ( E ) Cell contact area under control condition and PP2 treatment. Boxes, 1st and 3rd quartiles; whiskers, 10th and 90th percentiles; total 50 cells. (Scale bars, 5 μm).
    Figure Legend Snippet: Outward movement of integrin clusters follows protrusion of cell edges. ( A ) Long-ranged outward then inward translocation of ligated integrin complexes. ( B ) Preincubation of 10 μM PP2, a Src-kinase inhibitor, blocked outward movement and cell spreading. Inset overlay: bright-field images, respectively. Kymographs: lateral movement of marked clusters, respectively. ( C , D ) Radial positions of each cluster’s trajectory (colored aster) and averaged cell edges (yellow triangle) of (A, 27 clusters) and (B, 8 clusters). ( E ) Cell contact area under control condition and PP2 treatment. Boxes, 1st and 3rd quartiles; whiskers, 10th and 90th percentiles; total 50 cells. (Scale bars, 5 μm).

    Techniques Used: Translocation Assay

    27) Product Images from "Inhibition of Lck enhances glucocorticoid sensitivity and apoptosis in lymphoid cell lines and in chronic lymphocytic leukemia"

    Article Title: Inhibition of Lck enhances glucocorticoid sensitivity and apoptosis in lymphoid cell lines and in chronic lymphocytic leukemia

    Journal: Cell death and differentiation

    doi: 10.1038/cdd.2010.25

    Dasatinib enhances glucocorticoid sensitivity and apoptosis in primary CLL cells. ( a and b ) PBLs from CLL2 were treated with vehicle (0.1% ethanol) or 10 −6 M dexamethasone or with vehicles (0.1% ethanol and 0.01% DMSO), dexamethasone, 100 nM dasatinib or both for 24 h. Total Lck and phospho-Lck (Y394) were measured by western blotting (note that the antibody used to detect phospho-Lck may also crossreact with phospho-Lyn). β -Actin was used as a loading control. ( c ) PBLs from CLL2 were treated with vehicle or dexamethasone (10 −6 M) for 3, 6, and 24 h and GR-responsive protein Txnip was measured by western blotting to control for glucocorticoid uptake and GR functionality. ( d ) CLL cells and PBLs from a patient with leukemic mantle cell lymphoma were incubated in media containing vehicles (0.1% ethanol and 0.01% DMSO), dexamethasone, 100 nM dasatinib, or both for 24 h. Values represent the mean from treated and control samples. Statistical significance was determined by Student’s t -test. ( e ) PBLs from CLL14 were treated as in d with 10 −6 M dexamethasone for 72 h. ( f ) MEC1 cells were treated as in d for 24 h. ( g ) PBLs from CLL6 were treated as in d with 10 −6 M dexamethasone for 24 h. Apoptosis was determined by flow cytometric analysis of Annexin V and propidium iodide-stained cells. Control experiments are represented in blue dots. Experimental treatments are overlaid in red dots. ( h ) PBLs from CLL23 were treated with 10 −6 M dexamethasone and 10 μ M PP2 for 72 h. ( i ) PBLs from CLL5 were treated with vehicles (0.1% ethanol and 0.005% DMSO), 10 −6 M dexamethasone, 50 nM BIBF 1120, or both for 24 h. Cells were also treated with 0.1% ethanol or 10 −6 M dexamethasone in the presence of either nonphosphorylated (control) EGQYEEIP or phosphorylated EGQY*EEIP H 2 O soluble peptides (200 nM) for 24 h. Cell death was measured in triplicate by trypan blue dye exclusion. ( e ) Data are representative of multiple independent experiments. Error bars represent the S.E.M.
    Figure Legend Snippet: Dasatinib enhances glucocorticoid sensitivity and apoptosis in primary CLL cells. ( a and b ) PBLs from CLL2 were treated with vehicle (0.1% ethanol) or 10 −6 M dexamethasone or with vehicles (0.1% ethanol and 0.01% DMSO), dexamethasone, 100 nM dasatinib or both for 24 h. Total Lck and phospho-Lck (Y394) were measured by western blotting (note that the antibody used to detect phospho-Lck may also crossreact with phospho-Lyn). β -Actin was used as a loading control. ( c ) PBLs from CLL2 were treated with vehicle or dexamethasone (10 −6 M) for 3, 6, and 24 h and GR-responsive protein Txnip was measured by western blotting to control for glucocorticoid uptake and GR functionality. ( d ) CLL cells and PBLs from a patient with leukemic mantle cell lymphoma were incubated in media containing vehicles (0.1% ethanol and 0.01% DMSO), dexamethasone, 100 nM dasatinib, or both for 24 h. Values represent the mean from treated and control samples. Statistical significance was determined by Student’s t -test. ( e ) PBLs from CLL14 were treated as in d with 10 −6 M dexamethasone for 72 h. ( f ) MEC1 cells were treated as in d for 24 h. ( g ) PBLs from CLL6 were treated as in d with 10 −6 M dexamethasone for 24 h. Apoptosis was determined by flow cytometric analysis of Annexin V and propidium iodide-stained cells. Control experiments are represented in blue dots. Experimental treatments are overlaid in red dots. ( h ) PBLs from CLL23 were treated with 10 −6 M dexamethasone and 10 μ M PP2 for 72 h. ( i ) PBLs from CLL5 were treated with vehicles (0.1% ethanol and 0.005% DMSO), 10 −6 M dexamethasone, 50 nM BIBF 1120, or both for 24 h. Cells were also treated with 0.1% ethanol or 10 −6 M dexamethasone in the presence of either nonphosphorylated (control) EGQYEEIP or phosphorylated EGQY*EEIP H 2 O soluble peptides (200 nM) for 24 h. Cell death was measured in triplicate by trypan blue dye exclusion. ( e ) Data are representative of multiple independent experiments. Error bars represent the S.E.M.

    Techniques Used: Western Blot, Incubation, Flow Cytometry, Staining

    28) Product Images from "Haemophilus ducreyi LspA Proteins Are Tyrosine Phosphorylated by Macrophage-Encoded Protein Tyrosine Kinases ▿"

    Article Title: Haemophilus ducreyi LspA Proteins Are Tyrosine Phosphorylated by Macrophage-Encoded Protein Tyrosine Kinases ▿

    Journal:

    doi: 10.1128/IAI.00513-08

    Tyrosine phosphorylation of LpsA1 fusion proteins by purified c-Src PTK in vitro. (A) Purified HL8 was incubated with purified c-Src PTK under the following conditions: no enzyme (No Enz), buffer (Buf), DMSO, 10 μM PP2 (lane 4), or 100 μM
    Figure Legend Snippet: Tyrosine phosphorylation of LpsA1 fusion proteins by purified c-Src PTK in vitro. (A) Purified HL8 was incubated with purified c-Src PTK under the following conditions: no enzyme (No Enz), buffer (Buf), DMSO, 10 μM PP2 (lane 4), or 100 μM

    Techniques Used: Purification, In Vitro, Incubation

    Effect of the Src family PTK inhibitor PP2 on tyrosine phosphorylation of the LspA proteins by macrophages. Wild-type H. ducreyi 35000HP bacteria were incubated with J774A.1 macrophages in the presence of PBS, DMSO (solvent for PP2), 10 μM PP2
    Figure Legend Snippet: Effect of the Src family PTK inhibitor PP2 on tyrosine phosphorylation of the LspA proteins by macrophages. Wild-type H. ducreyi 35000HP bacteria were incubated with J774A.1 macrophages in the presence of PBS, DMSO (solvent for PP2), 10 μM PP2

    Techniques Used: Incubation

    Tyrosine phosphorylation of the HL8 fusion protein by macrophage lysates is inhibited by PP2. J774A.1 macrophage lysates were incubated with PBS or with HL8 with 100 μM PP2 (+) or without PP2 (−) for 60 min. No exogenous ATP was
    Figure Legend Snippet: Tyrosine phosphorylation of the HL8 fusion protein by macrophage lysates is inhibited by PP2. J774A.1 macrophage lysates were incubated with PBS or with HL8 with 100 μM PP2 (+) or without PP2 (−) for 60 min. No exogenous ATP was

    Techniques Used: Incubation

    29) Product Images from "Gα13 Stimulates the Tyrosine Phosphorylation of Ric-8A"

    Article Title: Gα13 Stimulates the Tyrosine Phosphorylation of Ric-8A

    Journal: Journal of Molecular Signaling

    doi: 10.5334/1750-2187-10-3

    Gα 13 induces Src-dependent plasma membrane translocation of Ric-8A. A. GFP or GFP-Ric-8A fusion construct was coexpressed with vectors expressing Gα 13 along with appropriate control vectors (1 mg) in COS-7 cells (1.5 × 10 6 cells/dish). At 48 hrs, the transfectants were lysed and the lysates were subjected to immunoblot (IB) analysis using the respective antibodies to monitor the expressions of GFP, GFP-Ric-8A and Gα 13 proteins. B. Cells transiently expressing GFP or GFP-Ric-8A fusion protein along with vectors expressing Gα 13 or vector control for 48 hrs were imaged by fluorescence microscopy. Localization of GFP-Ric-8A on plasma membrane (arrowheads) was observed in the presence of Gα 13 . Scale bar, 10 mm. C. COS7 cells transfected with GFP-Ric-8A and Gα 13 for 48 hrs were treated 10mM Src kinase inhibitor SI1 or PP2 for 16 hrs and the fluorescence of GFP was imaged. Plasma membrane localization (arrowheads) was attenuated in the presence of SI1 or PP2. Scale bar, 10 mm. Data are representative of three independent experiments with similar results.
    Figure Legend Snippet: Gα 13 induces Src-dependent plasma membrane translocation of Ric-8A. A. GFP or GFP-Ric-8A fusion construct was coexpressed with vectors expressing Gα 13 along with appropriate control vectors (1 mg) in COS-7 cells (1.5 × 10 6 cells/dish). At 48 hrs, the transfectants were lysed and the lysates were subjected to immunoblot (IB) analysis using the respective antibodies to monitor the expressions of GFP, GFP-Ric-8A and Gα 13 proteins. B. Cells transiently expressing GFP or GFP-Ric-8A fusion protein along with vectors expressing Gα 13 or vector control for 48 hrs were imaged by fluorescence microscopy. Localization of GFP-Ric-8A on plasma membrane (arrowheads) was observed in the presence of Gα 13 . Scale bar, 10 mm. C. COS7 cells transfected with GFP-Ric-8A and Gα 13 for 48 hrs were treated 10mM Src kinase inhibitor SI1 or PP2 for 16 hrs and the fluorescence of GFP was imaged. Plasma membrane localization (arrowheads) was attenuated in the presence of SI1 or PP2. Scale bar, 10 mm. Data are representative of three independent experiments with similar results.

    Techniques Used: Translocation Assay, Construct, Expressing, Plasmid Preparation, Fluorescence, Microscopy, Transfection

    30) Product Images from "Insulin-like growth factor binding protein-2 regulates β-catenin signaling pathway in glioma cells and contributes to poor patient prognosis"

    Article Title: Insulin-like growth factor binding protein-2 regulates β-catenin signaling pathway in glioma cells and contributes to poor patient prognosis

    Journal: Neuro-Oncology

    doi: 10.1093/neuonc/now053

    IGFBP-2-induced β-catenin activity was integrin-signaling dependent but IGF1R signaling-independent (A) Western blots showing regulation of FAK at Tyr925, GSK3β at Ser9 and total β-catenin in IGFBP-2 overexpressing LN229 and U87 cell lines in presence or absence of PP2. PP3 was used as a negative control for PP2. (B) Regulation of p-GSK3β when cells were treated with 500 ng/mL of IGFBP-2 in presence of PP2 or IGF1R inhibitor. (C) Top/flash luciferase assay indicating the activity of nuclear β-catenin in IGFBP-2 overexpressing cells when treated with PP2/PP3/RGD/Wnt3a/LiCl/IGF1R inhibitor ( n = 3, P
    Figure Legend Snippet: IGFBP-2-induced β-catenin activity was integrin-signaling dependent but IGF1R signaling-independent (A) Western blots showing regulation of FAK at Tyr925, GSK3β at Ser9 and total β-catenin in IGFBP-2 overexpressing LN229 and U87 cell lines in presence or absence of PP2. PP3 was used as a negative control for PP2. (B) Regulation of p-GSK3β when cells were treated with 500 ng/mL of IGFBP-2 in presence of PP2 or IGF1R inhibitor. (C) Top/flash luciferase assay indicating the activity of nuclear β-catenin in IGFBP-2 overexpressing cells when treated with PP2/PP3/RGD/Wnt3a/LiCl/IGF1R inhibitor ( n = 3, P

    Techniques Used: Activity Assay, Western Blot, Negative Control, Luciferase

    31) Product Images from "Formin-mediated actin polymerization at cell–cell junctions stabilizes E-cadherin and maintains monolayer integrity during wound repair"

    Article Title: Formin-mediated actin polymerization at cell–cell junctions stabilizes E-cadherin and maintains monolayer integrity during wound repair

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E16-06-0429

    Src kinase and Cdc42 are upstream activators of formin activity at the AJ. (A) Src kinase inhibition via PP2 treatment or siRNA-mediated KD phenocopies mDia1 or Fmnl3 KD. (B) PCR analysis for efficiency of Src KD. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as control. (C) Dispase assay for PP2-treated monolayers. Three independent experiments, with mean ± SD. (D) Activation of endogenous mDia1 via DAD expression (mVenus fluorescence in inset) rescues the Src-inhibition phenotype. Note the dramatic augmentation of the AJ in transfected cells in comparison to nontransfected neighbors. (E) Quantification of E-cadherin fluorescence intensity at the junction for D. Control monolayers were transfected with a GFP vector; ≥30 junctions from transfected cells for each condition from three experiments, with mean ± SEM. (F) Full-length Src-GFP localizes to the AJ, resulting in elevated levels of F-actin and E-cadherin (arrowheads, top). Src-GFP expression combined with Fmnl3 KD abrogates junctional F-actin and E-cadherin augmentation (bottom). (G, H) Quantification of F-actin and E-cadherin fluorescence intensities for F; ≥35 junctions from transfected cells for each condition from three experiments, with mean ± SEM. (I) Expression of constitutively active Cdc42 (GFP shown in inset) rescues the Src-inhibition phenotype. Note the restoration of lateral junctions (white arrowheads) in transfected cells vs. nontransfected neighbors. (J) Quantification of E-cadherin fluorescence intensity for I. Control monolayers were transfected with a GFP vector; ≥ 28 junctions from transfected cells for each condition from three experiments, with mean ± SEM. (K) Fmnl3-GFP (I111D) does not localize to AJ, with no effect on F-actin or E-cadherin. (L, M) Quantification of F-actin and E-cadherin fluorescence intensities for K; ≥26 junctions from transfected cells per condition from three experiments, with mean ± SEM. Statistical significance assessed using one-way ANOVA in C, E, G, H, and J; Student’s t test in L and M. Scale bars, 20 μm (A, D, F, and K), 10 μm (I).
    Figure Legend Snippet: Src kinase and Cdc42 are upstream activators of formin activity at the AJ. (A) Src kinase inhibition via PP2 treatment or siRNA-mediated KD phenocopies mDia1 or Fmnl3 KD. (B) PCR analysis for efficiency of Src KD. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as control. (C) Dispase assay for PP2-treated monolayers. Three independent experiments, with mean ± SD. (D) Activation of endogenous mDia1 via DAD expression (mVenus fluorescence in inset) rescues the Src-inhibition phenotype. Note the dramatic augmentation of the AJ in transfected cells in comparison to nontransfected neighbors. (E) Quantification of E-cadherin fluorescence intensity at the junction for D. Control monolayers were transfected with a GFP vector; ≥30 junctions from transfected cells for each condition from three experiments, with mean ± SEM. (F) Full-length Src-GFP localizes to the AJ, resulting in elevated levels of F-actin and E-cadherin (arrowheads, top). Src-GFP expression combined with Fmnl3 KD abrogates junctional F-actin and E-cadherin augmentation (bottom). (G, H) Quantification of F-actin and E-cadherin fluorescence intensities for F; ≥35 junctions from transfected cells for each condition from three experiments, with mean ± SEM. (I) Expression of constitutively active Cdc42 (GFP shown in inset) rescues the Src-inhibition phenotype. Note the restoration of lateral junctions (white arrowheads) in transfected cells vs. nontransfected neighbors. (J) Quantification of E-cadherin fluorescence intensity for I. Control monolayers were transfected with a GFP vector; ≥ 28 junctions from transfected cells for each condition from three experiments, with mean ± SEM. (K) Fmnl3-GFP (I111D) does not localize to AJ, with no effect on F-actin or E-cadherin. (L, M) Quantification of F-actin and E-cadherin fluorescence intensities for K; ≥26 junctions from transfected cells per condition from three experiments, with mean ± SEM. Statistical significance assessed using one-way ANOVA in C, E, G, H, and J; Student’s t test in L and M. Scale bars, 20 μm (A, D, F, and K), 10 μm (I).

    Techniques Used: Activity Assay, Inhibition, Polymerase Chain Reaction, Activation Assay, Expressing, Fluorescence, Transfection, Plasmid Preparation

    32) Product Images from "Murine macrophage TLR2-FcγR synergy via FcγR licensing of IL-6 cytokine mRNA ribosome binding and translation"

    Article Title: Murine macrophage TLR2-FcγR synergy via FcγR licensing of IL-6 cytokine mRNA ribosome binding and translation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0200764

    mAb-iB TLR2 induction of IL-6 mRNA requires TLR2 but not FcγR ITAM signaling. BMMØ were stimulated with mAb-iB TLR2 ) and the level of secreted IL-6 or TNF protein determined by ELISA (ND = not detected). The mean level of cytokine detected in cultures supernatants from control mAb-iB TLR2 stimulated BMMØ was 554 pg/m of IL-6 (panel A), 776 pg/ml of IL-6 (panel B) and 670 pg/ml of TNF (panel C). Show are the average levels of cytokine mRNA and protein (normalized to control non-PP2 WT BMMØ) across three or more independent experiments (error bars = ± 1 S.D.). ** = p
    Figure Legend Snippet: mAb-iB TLR2 induction of IL-6 mRNA requires TLR2 but not FcγR ITAM signaling. BMMØ were stimulated with mAb-iB TLR2 ) and the level of secreted IL-6 or TNF protein determined by ELISA (ND = not detected). The mean level of cytokine detected in cultures supernatants from control mAb-iB TLR2 stimulated BMMØ was 554 pg/m of IL-6 (panel A), 776 pg/ml of IL-6 (panel B) and 670 pg/ml of TNF (panel C). Show are the average levels of cytokine mRNA and protein (normalized to control non-PP2 WT BMMØ) across three or more independent experiments (error bars = ± 1 S.D.). ** = p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    33) Product Images from "ER-?36-mediated gastric cancer cell proliferation via the c-Src pathway"

    Article Title: ER-?36-mediated gastric cancer cell proliferation via the c-Src pathway

    Journal: Oncology Letters

    doi: 10.3892/ol.2013.1416

    Estrogen induces cyclin D1 expression through activation of the ER-α36 pathway. (A) Western blot analysis of cyclin D1 expression in SGC7901, High36 and Low36 cell lines. Cells were treated with E2β alone. (B) Western blot analysis of cyclin D1 expression in SGC7901, High36 and Low36 cells. Cells were treated with the c-Src inhibitor, PP2 and E2β. (C) Western blot analysis of cyclin D1 expression in SGC7901, High36 and Low36 cells. Cells were treated with the PP2 c-Src inhibitor alone. (D) Estrogen induces cyclin D1 expression through activation of the ER-α36 pathway and the PP2 c-Src inhibitor downregulates cyclin D1 expression in gastric cancer cells ( * P
    Figure Legend Snippet: Estrogen induces cyclin D1 expression through activation of the ER-α36 pathway. (A) Western blot analysis of cyclin D1 expression in SGC7901, High36 and Low36 cell lines. Cells were treated with E2β alone. (B) Western blot analysis of cyclin D1 expression in SGC7901, High36 and Low36 cells. Cells were treated with the c-Src inhibitor, PP2 and E2β. (C) Western blot analysis of cyclin D1 expression in SGC7901, High36 and Low36 cells. Cells were treated with the PP2 c-Src inhibitor alone. (D) Estrogen induces cyclin D1 expression through activation of the ER-α36 pathway and the PP2 c-Src inhibitor downregulates cyclin D1 expression in gastric cancer cells ( * P

    Techniques Used: Expressing, Activation Assay, Western Blot

    ER-α36-c-Src interaction analysis. Extracts from SGC7901 cells were incubated with E2β and/or with PP2. Formed complexes were pulled down and analyzed using antibodies against (A) c-Src and ER-α36, (B) p416-c-Src and ER-α36 and (C) p527-c-Src and ER-α36. ER, estrogen receptor; E2β, 17β-estradiol.
    Figure Legend Snippet: ER-α36-c-Src interaction analysis. Extracts from SGC7901 cells were incubated with E2β and/or with PP2. Formed complexes were pulled down and analyzed using antibodies against (A) c-Src and ER-α36, (B) p416-c-Src and ER-α36 and (C) p527-c-Src and ER-α36. ER, estrogen receptor; E2β, 17β-estradiol.

    Techniques Used: Incubation

    PP2 inhibits the activation of c-Src induced by E2β. (A) PP2 blocks c-Src activation in SGC7901, High36 and Low36 cell lines from days 5–11 and PP2 reduced proliferation in ∼68.91 and 91.56% of High36 and Low36 cells, respectively, at day 11. (B) E2β and/or PP2-stimulated SGC7901, High36 and Low36 cells at 0, 5 and 20 min. Cell lysates were analyzed with anti-p416-c-Src and anti-p527-c-Src antibodies. Anti-c-Src antibody was used to ensure equal loading. Western blot analysis of p-416-c-Src and p-527-c-Src expression in SGC7901, High36 and Low36 cells. E2β, 17β-estradiol.
    Figure Legend Snippet: PP2 inhibits the activation of c-Src induced by E2β. (A) PP2 blocks c-Src activation in SGC7901, High36 and Low36 cell lines from days 5–11 and PP2 reduced proliferation in ∼68.91 and 91.56% of High36 and Low36 cells, respectively, at day 11. (B) E2β and/or PP2-stimulated SGC7901, High36 and Low36 cells at 0, 5 and 20 min. Cell lysates were analyzed with anti-p416-c-Src and anti-p527-c-Src antibodies. Anti-c-Src antibody was used to ensure equal loading. Western blot analysis of p-416-c-Src and p-527-c-Src expression in SGC7901, High36 and Low36 cells. E2β, 17β-estradiol.

    Techniques Used: Activation Assay, Western Blot, Expressing

    34) Product Images from "Integrin-based diffusion barrier separates membrane domains enabling the formation of microbiostatic frustrated phagosomes"

    Article Title: Integrin-based diffusion barrier separates membrane domains enabling the formation of microbiostatic frustrated phagosomes

    Journal: eLife

    doi: 10.7554/eLife.34798

    Signaling associated with actin polymerization at the phagocytic cup formed around C. albicans hyphae. After incubation with Candida -BFP hyphae, RAW-Dectin1 cells were fixed and extracellular C. albicans stained using fluorescent concanavalin A. ( A ) Phosphotyrosine (pTyrosine) was detected by immunostaining (green). F-actin was visualized using TdTom-F-Tractin (red); concanavalin A (blue). Inset: Colocalization of actin cuff with pTyrosine, in yellow. Image is representative of ≥30 fields from ≥3 separate experiments. ( B ) Phospho-SFK (Y418) was detected by immunostaining (green); concanavalin A (red). Inset: Colocalization of actin cuff with pSFK, in yellow. ( C ) Phospho-PYK2 (Y402) was detected by immunostaining (green); concanavalin A (red). Inset: Colocalization of actin cuff with pPYK2, in yellow. ( D ) Phospho-FAK (Y397) was detected by immunostaining (green); concanavalin A (red). Inset: Colocalization of actin cuff with pFAK, in yellow. Images in B, C and D are representative of ≥30 fields from ≥2 separate experiments of each type. ( E ) Effect of tyrosine kinase inhibitors on actin cuff formation. RAW-Dectin1 cells were allowed to adhere Candida -BFP hyphae for 15 min and then incubated 45 min in the presence of vehicle, PP2, piceatannol or PF573228. Following phagocytosis, extracellular C. albicans was stained using concanavalin A, and actin stained with phalloidin. The number of C. albicans hyphae that were fully internalized or partially internalized with actin cuffs per 94.5x field was counted by confocal microscopy. Average number of C. albicans per field was 3.4 ± 0.6. For each condition, three independent experiments were quantified, with ≥4 fields counted per replicate. p value was calculated using unpaired, 2-tailed students t-test. Data are means ±SEM. ( F ) Active Rac/Cdc42 were visualized using PAK(PBD)-YFP as a probe (green). Actin was stained using fluorescent phalloidin (blue); concanavalin A (red). Inset: Colocalization of actin cuff with PAK(PBD), in yellow. Image is representative of ≥30 fields from ≥3 separate experiments. Scale bars: 10 μm. ( G ) Effect of actin assembly inhibitors on actin cuff formation. RAW-Dectin1 cells were allowed to adhere Candida -BFP hyphae for 15 min, then incubated 45 min in the presence of vehicle, CK-666 or SMI-FH2. Following phagocytosis, extracellular C. albicans was stained using concanavalin A, and actin stained with phalloidin. The number of C. albicans hyphae that were fully internalized or partially internalized with actin cuffs per 94.5x field was counted by confocal microscopy. Average number of C. albicans per field as in (E). For each condition, three independent experiments were quantified, with ≥5 fields counted per replicate. p value was calculated using the unpaired, 2-tailed students t-test. Data are means ±SEM.
    Figure Legend Snippet: Signaling associated with actin polymerization at the phagocytic cup formed around C. albicans hyphae. After incubation with Candida -BFP hyphae, RAW-Dectin1 cells were fixed and extracellular C. albicans stained using fluorescent concanavalin A. ( A ) Phosphotyrosine (pTyrosine) was detected by immunostaining (green). F-actin was visualized using TdTom-F-Tractin (red); concanavalin A (blue). Inset: Colocalization of actin cuff with pTyrosine, in yellow. Image is representative of ≥30 fields from ≥3 separate experiments. ( B ) Phospho-SFK (Y418) was detected by immunostaining (green); concanavalin A (red). Inset: Colocalization of actin cuff with pSFK, in yellow. ( C ) Phospho-PYK2 (Y402) was detected by immunostaining (green); concanavalin A (red). Inset: Colocalization of actin cuff with pPYK2, in yellow. ( D ) Phospho-FAK (Y397) was detected by immunostaining (green); concanavalin A (red). Inset: Colocalization of actin cuff with pFAK, in yellow. Images in B, C and D are representative of ≥30 fields from ≥2 separate experiments of each type. ( E ) Effect of tyrosine kinase inhibitors on actin cuff formation. RAW-Dectin1 cells were allowed to adhere Candida -BFP hyphae for 15 min and then incubated 45 min in the presence of vehicle, PP2, piceatannol or PF573228. Following phagocytosis, extracellular C. albicans was stained using concanavalin A, and actin stained with phalloidin. The number of C. albicans hyphae that were fully internalized or partially internalized with actin cuffs per 94.5x field was counted by confocal microscopy. Average number of C. albicans per field was 3.4 ± 0.6. For each condition, three independent experiments were quantified, with ≥4 fields counted per replicate. p value was calculated using unpaired, 2-tailed students t-test. Data are means ±SEM. ( F ) Active Rac/Cdc42 were visualized using PAK(PBD)-YFP as a probe (green). Actin was stained using fluorescent phalloidin (blue); concanavalin A (red). Inset: Colocalization of actin cuff with PAK(PBD), in yellow. Image is representative of ≥30 fields from ≥3 separate experiments. Scale bars: 10 μm. ( G ) Effect of actin assembly inhibitors on actin cuff formation. RAW-Dectin1 cells were allowed to adhere Candida -BFP hyphae for 15 min, then incubated 45 min in the presence of vehicle, CK-666 or SMI-FH2. Following phagocytosis, extracellular C. albicans was stained using concanavalin A, and actin stained with phalloidin. The number of C. albicans hyphae that were fully internalized or partially internalized with actin cuffs per 94.5x field was counted by confocal microscopy. Average number of C. albicans per field as in (E). For each condition, three independent experiments were quantified, with ≥5 fields counted per replicate. p value was calculated using the unpaired, 2-tailed students t-test. Data are means ±SEM.

    Techniques Used: Incubation, Staining, Immunostaining, Confocal Microscopy

    35) Product Images from "Pseudomonas aeruginosa interacts with epithelial cells rapidly forming aggregates that are internalized by a Lyn-dependent mechanism"

    Article Title: Pseudomonas aeruginosa interacts with epithelial cells rapidly forming aggregates that are internalized by a Lyn-dependent mechanism

    Journal: Cellular microbiology

    doi: 10.1111/j.1462-5822.2011.01611.x

    Entry of P. aeruginosa aggregates is dependent on Src family tyrosine kinases. GFP-PH-Akt MDCK cells were infected with m-Cherry P. aeruginosa for 2 h. Treatment with SFKs inhibitor PP2 (10 µm) reduced the percentage of internalized individual
    Figure Legend Snippet: Entry of P. aeruginosa aggregates is dependent on Src family tyrosine kinases. GFP-PH-Akt MDCK cells were infected with m-Cherry P. aeruginosa for 2 h. Treatment with SFKs inhibitor PP2 (10 µm) reduced the percentage of internalized individual

    Techniques Used: Infection

    36) Product Images from "BDCA-2, a Novel Plasmacytoid Dendritic Cell-specific Type II C-type Lectin, Mediates Antigen Capture and Is a Potent Inhibitor of Interferon ?/? Induction"

    Article Title: BDCA-2, a Novel Plasmacytoid Dendritic Cell-specific Type II C-type Lectin, Mediates Antigen Capture and Is a Potent Inhibitor of Interferon ?/? Induction

    Journal: The Journal of Experimental Medicine

    doi:

    A rapid and transient rise in [Ca 2+ ]i is induced in PDCs (left dotplots) and BDCA-2-transfected U937 cells (middle dotplots), but not in nontransfected U937 cells (right dotplots) after ligation of surface BDCA-2 with specific primary mAb (AC144, IgG1) and secondary cross-linking F(ab′) 2 goat anti–mouse IgG (B). This [Ca 2+ ]i increase is not affected when extracellular calcium is chelated with excess EGTA (C), but inhibited when src-family protein-tyrosine kinases are blocked by preincubation with the specific inhibitor PP2 (D). One representative experiment of six is shown.
    Figure Legend Snippet: A rapid and transient rise in [Ca 2+ ]i is induced in PDCs (left dotplots) and BDCA-2-transfected U937 cells (middle dotplots), but not in nontransfected U937 cells (right dotplots) after ligation of surface BDCA-2 with specific primary mAb (AC144, IgG1) and secondary cross-linking F(ab′) 2 goat anti–mouse IgG (B). This [Ca 2+ ]i increase is not affected when extracellular calcium is chelated with excess EGTA (C), but inhibited when src-family protein-tyrosine kinases are blocked by preincubation with the specific inhibitor PP2 (D). One representative experiment of six is shown.

    Techniques Used: Transfection, Ligation

    37) Product Images from "Unconjugated secondary bile acids activate the unfolded protein response and induce golgi fragmentation via a src-kinase-dependant mechanism"

    Article Title: Unconjugated secondary bile acids activate the unfolded protein response and induce golgi fragmentation via a src-kinase-dependant mechanism

    Journal: Oncotarget

    doi: 10.18632/oncotarget.13514

    Unconjugated bile acids cause Golgi fragmentation and Bip activation via activation of Src A. HET-1A cells were treated with Bile acids and phosphorylation of Src was measured by western blot. B. Cells were pre-treated with PP2 (10 μM), prior to treatment with bile acids. Golgi structure was assessed using a GM130 antibody (green) and imaged using confocal microscopy as outlined in materials and methods. Nuclei were stained with Hoechst (blue). C. HET-1A cells were pre-treated with PP2 (10 μM) followed by treatment with DCA. Levels of Bip mRNA were quantified by RT-PCR. Data are represented as mean ± SEM, normalised to vehicle control for n=3 experiments, * p
    Figure Legend Snippet: Unconjugated bile acids cause Golgi fragmentation and Bip activation via activation of Src A. HET-1A cells were treated with Bile acids and phosphorylation of Src was measured by western blot. B. Cells were pre-treated with PP2 (10 μM), prior to treatment with bile acids. Golgi structure was assessed using a GM130 antibody (green) and imaged using confocal microscopy as outlined in materials and methods. Nuclei were stained with Hoechst (blue). C. HET-1A cells were pre-treated with PP2 (10 μM) followed by treatment with DCA. Levels of Bip mRNA were quantified by RT-PCR. Data are represented as mean ± SEM, normalised to vehicle control for n=3 experiments, * p

    Techniques Used: Activation Assay, Western Blot, Confocal Microscopy, Staining, Reverse Transcription Polymerase Chain Reaction

    38) Product Images from "G6b-B Inhibits Constitutive and Agonist-induced Signaling by Glycoprotein VI and CLEC-2 *G6b-B Inhibits Constitutive and Agonist-induced Signaling by Glycoprotein VI and CLEC-2 * S⃞"

    Article Title: G6b-B Inhibits Constitutive and Agonist-induced Signaling by Glycoprotein VI and CLEC-2 *G6b-B Inhibits Constitutive and Agonist-induced Signaling by Glycoprotein VI and CLEC-2 * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M806895200

    Constitutive tyrosine phosphorylation of FcR γ-chain and Syk in human platelets. A , human washed platelets were incubated at 37 °C with the Syk kinase inhibitor, R406 (1 μ m ), and Src inhibitor PP2 (20 μ m ) for 1 min. Whole cell lysates were probed for phosphotyrosine ( pTyr ). B , wild-type ( WT ) or FcR γ-chain deficient mouse ( KO ) platelets (pretreated with EGTA to block aggregation) were stimulated with 100 μ m pervanadate ( PV ) for 90 s in the presence or absence of Src inhibitor PP2 (20 μ m ). C , human washed platelets were incubated at 37 °C with the Src kinase inhibitors PP1 (10 μ m ) or PD0173952 (10 μ m ) for 5 min. Cell lysates were immunoprecipitated with anti-Syk Ab or an IgG and probed for Tyr(P). Subsequently, they were stripped and reprobed for Syk. D , human washed platelets were stimulated with 30 n m rhodocytin for 5 min in the presence and absence of the Src kinase inhibitor, PP2 (10 μ m ). Cell lysates were immunoprecipitated ( IP ) with anti-CLEC-2 Ab and probed for Tyr(P). Subsequently, they were stripped and reprobed for CLEC-2. The results are representative of between 3 and 8 experiments. WB , Western blot.
    Figure Legend Snippet: Constitutive tyrosine phosphorylation of FcR γ-chain and Syk in human platelets. A , human washed platelets were incubated at 37 °C with the Syk kinase inhibitor, R406 (1 μ m ), and Src inhibitor PP2 (20 μ m ) for 1 min. Whole cell lysates were probed for phosphotyrosine ( pTyr ). B , wild-type ( WT ) or FcR γ-chain deficient mouse ( KO ) platelets (pretreated with EGTA to block aggregation) were stimulated with 100 μ m pervanadate ( PV ) for 90 s in the presence or absence of Src inhibitor PP2 (20 μ m ). C , human washed platelets were incubated at 37 °C with the Src kinase inhibitors PP1 (10 μ m ) or PD0173952 (10 μ m ) for 5 min. Cell lysates were immunoprecipitated with anti-Syk Ab or an IgG and probed for Tyr(P). Subsequently, they were stripped and reprobed for Syk. D , human washed platelets were stimulated with 30 n m rhodocytin for 5 min in the presence and absence of the Src kinase inhibitor, PP2 (10 μ m ). Cell lysates were immunoprecipitated ( IP ) with anti-CLEC-2 Ab and probed for Tyr(P). Subsequently, they were stripped and reprobed for CLEC-2. The results are representative of between 3 and 8 experiments. WB , Western blot.

    Techniques Used: Incubation, Blocking Assay, Immunoprecipitation, Western Blot

    39) Product Images from "Estrogen utilization of IGF-1-R and EGF-R to signal in breast cancer cells"

    Article Title: Estrogen utilization of IGF-1-R and EGF-R to signal in breast cancer cells

    Journal: The Journal of steroid biochemistry and molecular biology

    doi: 10.1016/j.jsbmb.2009.09.018

    Biological effect of fulvestrant on reporter gene expression and MCF-7 cell growth. A : Fulvestrant exerts anti-estrogenic activities on genomic endpoints. MCF-7 cells were transiently transfected with the luciferase reporter genes driven by either ERE (left panel) or E2F1 (right panel) promoters for 2 days. Then cells were treated with vehicle, different doses of E2 and ICI as indicated for 9 hrs. The luciferase activities were measured using a luminometer. B and C : Blockade or knock-down of Src or IGF-1R potentiates fulvestrant anti-estrogenic activity. Cells were pretreated with 5 μM PP2 or 1 μM AG1024 for 30 min ( B ) or transiently transfected with empty or IGF-1R expression vectors for 2 days ( C ). Then cells were challenged with vehicle or ICI at 100 nM for 5 days. The cell number was counted. Data are mean ± SEM (n=6). * p
    Figure Legend Snippet: Biological effect of fulvestrant on reporter gene expression and MCF-7 cell growth. A : Fulvestrant exerts anti-estrogenic activities on genomic endpoints. MCF-7 cells were transiently transfected with the luciferase reporter genes driven by either ERE (left panel) or E2F1 (right panel) promoters for 2 days. Then cells were treated with vehicle, different doses of E2 and ICI as indicated for 9 hrs. The luciferase activities were measured using a luminometer. B and C : Blockade or knock-down of Src or IGF-1R potentiates fulvestrant anti-estrogenic activity. Cells were pretreated with 5 μM PP2 or 1 μM AG1024 for 30 min ( B ) or transiently transfected with empty or IGF-1R expression vectors for 2 days ( C ). Then cells were challenged with vehicle or ICI at 100 nM for 5 days. The cell number was counted. Data are mean ± SEM (n=6). * p

    Techniques Used: Expressing, Transfection, Luciferase, Activity Assay

    Fulvestrant at 100 nM activates both IGF-1R and MAPK in MCF-7 cells. A : Fulvestrant induced IGF-1R phosphorylation. Cells were treated with either vehicle, 10 ng/ml IGF-1 as positive control or 100 nM ICI for the time indicated and the IGF-1R phosphorylation was assessed by immunoprecipitation of IGF-1R and detection of its phosphorylation status using anti-pY antibody 4G10. B and C Fulvestrant-induced MAPK phosphorylation. Cells were treated with vehicle or fulvestrant at the doses indicated for 15 min (B) or 100 nM fulvestrant for the time indicated (C). The phosphorylation status and total protein loading of MAPK were assayed using specific anti-active and anti-MAPK antibodies. D : PP2 and AG1024 blocked fulvestrant-induced MAPK phosphorylation. Cells were pre-treated with 5 μM PP2 or 1 μM AG1024, and then challenged with vehicle or 100 nM fulvestrant for 15 min. The phosphorylation status and total MAPK protein loading were assayed using specific anti-active and anti-MAPK antibodies. E : Confocal microscopic study of fulvestrant-induced MAPK phosphorylation. Cells were starved in 1% DCC medium for 24 hr and treated with either vehicle (upper row), ICI at 100 nM (middle row) or IGF-1R at 10 ng/ml (bottom row) for 15 min. Treated cells were fixed in 4% formaldehyde, permeabilized with cold acetone, and stained with rabbit polyclonal anti-phosphor MAPK antibody (a, e, i), phalloidin to stain membrane filamentous actin (b, f, j) or nuclear staining with DAPI (c, g, k). The merged images are shown in d, h, l. Scale bar = 20 μm.
    Figure Legend Snippet: Fulvestrant at 100 nM activates both IGF-1R and MAPK in MCF-7 cells. A : Fulvestrant induced IGF-1R phosphorylation. Cells were treated with either vehicle, 10 ng/ml IGF-1 as positive control or 100 nM ICI for the time indicated and the IGF-1R phosphorylation was assessed by immunoprecipitation of IGF-1R and detection of its phosphorylation status using anti-pY antibody 4G10. B and C Fulvestrant-induced MAPK phosphorylation. Cells were treated with vehicle or fulvestrant at the doses indicated for 15 min (B) or 100 nM fulvestrant for the time indicated (C). The phosphorylation status and total protein loading of MAPK were assayed using specific anti-active and anti-MAPK antibodies. D : PP2 and AG1024 blocked fulvestrant-induced MAPK phosphorylation. Cells were pre-treated with 5 μM PP2 or 1 μM AG1024, and then challenged with vehicle or 100 nM fulvestrant for 15 min. The phosphorylation status and total MAPK protein loading were assayed using specific anti-active and anti-MAPK antibodies. E : Confocal microscopic study of fulvestrant-induced MAPK phosphorylation. Cells were starved in 1% DCC medium for 24 hr and treated with either vehicle (upper row), ICI at 100 nM (middle row) or IGF-1R at 10 ng/ml (bottom row) for 15 min. Treated cells were fixed in 4% formaldehyde, permeabilized with cold acetone, and stained with rabbit polyclonal anti-phosphor MAPK antibody (a, e, i), phalloidin to stain membrane filamentous actin (b, f, j) or nuclear staining with DAPI (c, g, k). The merged images are shown in d, h, l. Scale bar = 20 μm.

    Techniques Used: Positive Control, Immunoprecipitation, Droplet Countercurrent Chromatography, Staining

    Role of Src and IGF-1R in fulvestrant-induced ERα/IGF-1R interaction. A : Inhibitors of Src and IGF-1R tyrosine kinase blocked fulvestrant-induced ERα/IGF-1R interaction. Cells were pre-treated 30 min with 5 μM PP2, 1 μM AG1024, 1 μM AG1478 or 20 μM PD98059 (PD). Cells were then challenged with ICI at 100 nM for 15 min. The interaction of ERα and IGF-1R was assayed as described in Materials and Methods. B : Src expression altered fulvestrant-induced ERα/IGF-1R interaction. Cells were transiently transfected with non-specific scramble siRNA (siCont) or siRNA against Src (upper panel) or expression vector with or without insert of wild type Src (lower panel) for 3 days. Then cells were challenged with vehicle or ICI at 100 nM for 15 min. The interaction of ERα and IGF-1R was assayed as described in Materials and Methods. The Western blots (WB) in both upper and lower panels show either Src knockdown or over-expression in the cells. The MCF-7 cell extract loaded on Western blot to monitor protein migrations was used as a positive control (pc). C : Knock-down of IGF-1R abolished fulvestrant-induced ERα and IGF-1R association. Cells were transiently transfected with non-specific scramble siRNA (siCont) or siRNA against IGF-1R for 3 days, then cells were challenged with vehicle or ICI at 100 nM for 15 min. The interaction of ERα and IGF-1R was assayed as described in Materials and Methods. D : PP2 and AG0124 further potentiate the fulvestrant effect on cellular ERα degradation. Cells were pretreated with the selective inhibitors as indicated for 30 min and then challenged with vehicle or ICI at 100 nM for 3 hrs. The total ERα level was assayed by Western blot. The blot was also probed with actin to normalize protein loadings.
    Figure Legend Snippet: Role of Src and IGF-1R in fulvestrant-induced ERα/IGF-1R interaction. A : Inhibitors of Src and IGF-1R tyrosine kinase blocked fulvestrant-induced ERα/IGF-1R interaction. Cells were pre-treated 30 min with 5 μM PP2, 1 μM AG1024, 1 μM AG1478 or 20 μM PD98059 (PD). Cells were then challenged with ICI at 100 nM for 15 min. The interaction of ERα and IGF-1R was assayed as described in Materials and Methods. B : Src expression altered fulvestrant-induced ERα/IGF-1R interaction. Cells were transiently transfected with non-specific scramble siRNA (siCont) or siRNA against Src (upper panel) or expression vector with or without insert of wild type Src (lower panel) for 3 days. Then cells were challenged with vehicle or ICI at 100 nM for 15 min. The interaction of ERα and IGF-1R was assayed as described in Materials and Methods. The Western blots (WB) in both upper and lower panels show either Src knockdown or over-expression in the cells. The MCF-7 cell extract loaded on Western blot to monitor protein migrations was used as a positive control (pc). C : Knock-down of IGF-1R abolished fulvestrant-induced ERα and IGF-1R association. Cells were transiently transfected with non-specific scramble siRNA (siCont) or siRNA against IGF-1R for 3 days, then cells were challenged with vehicle or ICI at 100 nM for 15 min. The interaction of ERα and IGF-1R was assayed as described in Materials and Methods. D : PP2 and AG0124 further potentiate the fulvestrant effect on cellular ERα degradation. Cells were pretreated with the selective inhibitors as indicated for 30 min and then challenged with vehicle or ICI at 100 nM for 3 hrs. The total ERα level was assayed by Western blot. The blot was also probed with actin to normalize protein loadings.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, Over Expression, Positive Control

    40) Product Images from "Transforming growth factor‐β enhances Rho‐kinase activity and contraction in airway smooth muscle via the nucleotide exchange factor ARHGEF1"

    Article Title: Transforming growth factor‐β enhances Rho‐kinase activity and contraction in airway smooth muscle via the nucleotide exchange factor ARHGEF1

    Journal: The Journal of Physiology

    doi: 10.1113/JP275033

    Effects of TGF‐β and SrcFK/RhoGEF/Rho‐kinase inhibition on BK‐induced contraction Measurement of isometric tension in isolated rat bronchioles mounted on the wire myograph. A , representative traces showing bradykinin (BK) applied cumulatively (0.01–100 μ m ) at 5 min intervals, after 18 h pre‐incubation in media only or in media with 30 ng ml −1 TGF‐β. Arrows indicate when the first dose of BK was applied. B – E , mean contraction amplitude after TGF‐β or media pre‐incubation measured at the plateau phase of each BK application, in the absence ( B , media: n = 24, TGF‐β: n = 23) or presence of 30 μ m PP2 ( C , media: n = 17, TGF‐β: n = 15), 30 μ m Y16 ( D , media: n = 9, TGF‐β: n = 9) or 10 μ m Y27632 ( E , media: n = 9, TGF‐β: n = 9). F and G , non‐linear regression data, showing effects of TGF‐β and inhibitors on Bmax ( F ) and PD2 values ( G ). * P
    Figure Legend Snippet: Effects of TGF‐β and SrcFK/RhoGEF/Rho‐kinase inhibition on BK‐induced contraction Measurement of isometric tension in isolated rat bronchioles mounted on the wire myograph. A , representative traces showing bradykinin (BK) applied cumulatively (0.01–100 μ m ) at 5 min intervals, after 18 h pre‐incubation in media only or in media with 30 ng ml −1 TGF‐β. Arrows indicate when the first dose of BK was applied. B – E , mean contraction amplitude after TGF‐β or media pre‐incubation measured at the plateau phase of each BK application, in the absence ( B , media: n = 24, TGF‐β: n = 23) or presence of 30 μ m PP2 ( C , media: n = 17, TGF‐β: n = 15), 30 μ m Y16 ( D , media: n = 9, TGF‐β: n = 9) or 10 μ m Y27632 ( E , media: n = 9, TGF‐β: n = 9). F and G , non‐linear regression data, showing effects of TGF‐β and inhibitors on Bmax ( F ) and PD2 values ( G ). * P

    Techniques Used: Inhibition, Isolation, Incubation

    41) Product Images from "Caveolin-1 Associated Adenovirus Entry into Human Corneal Cells"

    Article Title: Caveolin-1 Associated Adenovirus Entry into Human Corneal Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0077462

    The role of caveolin-1 and Src kinase in HAdV entry and inflammation. A . C57BL/6J wild type (WT) and caveolin-1 -/- mice (on the C57BL/6J background) were injected intrastromally with Cy3-labeled (red) HAdV-D37 (10 5 tissue culture infectious doses) or buffer control, and the corneas harvested for confocal microscopy. Phalloidin (green) co-staining was used to better visualize the cells. Virus entry was apparent by 1 hr post infection in WT mice. In caveolin-1 -/- mice, virus remained mostly membrane bound in the first hour post infection and internalization appeared reduced at 24 hr compared to WT mice. B . Western blot analysis of the cornea from mice infected with HAdV-D37 revealed increasing pSrc at 1 and 24 hr post infection, while expression was minimal in untouched (uninfected) corneas. Caveolin-1 -/- mice did not show increased pSrc even after 24 hr infection. Actin controls are shown in the bottom panel. C . ELISA for CXCL1 chemokine performed on WT and caveolin-1 -/- mice corneas at 24 hr post infection with HAdV-D37. Infected caveolin-1 -/- mice corneas showed approximately 60% less CXCL1 expression as compared to infected wild type corneas (*p=.0001). Mock infected mice corneas did not produce any detectable CXCL1. D . Histology of PP2 or DMSO (control) pretreated corneas at 4 days post infection with HAdV-D37 or buffer control shows a reduction of keratitis with chemical inhibition of Src kinase. E . CXCL1 expression by ELISA of HAdV-D37 infected or mock infected corneas at 16 hr post infection pretreated with the Src kinase inhibitor PP2 (10 μM) or DMSO control demonstrates a significant reduction in chemokine expression due to Src inhibition (*p
    Figure Legend Snippet: The role of caveolin-1 and Src kinase in HAdV entry and inflammation. A . C57BL/6J wild type (WT) and caveolin-1 -/- mice (on the C57BL/6J background) were injected intrastromally with Cy3-labeled (red) HAdV-D37 (10 5 tissue culture infectious doses) or buffer control, and the corneas harvested for confocal microscopy. Phalloidin (green) co-staining was used to better visualize the cells. Virus entry was apparent by 1 hr post infection in WT mice. In caveolin-1 -/- mice, virus remained mostly membrane bound in the first hour post infection and internalization appeared reduced at 24 hr compared to WT mice. B . Western blot analysis of the cornea from mice infected with HAdV-D37 revealed increasing pSrc at 1 and 24 hr post infection, while expression was minimal in untouched (uninfected) corneas. Caveolin-1 -/- mice did not show increased pSrc even after 24 hr infection. Actin controls are shown in the bottom panel. C . ELISA for CXCL1 chemokine performed on WT and caveolin-1 -/- mice corneas at 24 hr post infection with HAdV-D37. Infected caveolin-1 -/- mice corneas showed approximately 60% less CXCL1 expression as compared to infected wild type corneas (*p=.0001). Mock infected mice corneas did not produce any detectable CXCL1. D . Histology of PP2 or DMSO (control) pretreated corneas at 4 days post infection with HAdV-D37 or buffer control shows a reduction of keratitis with chemical inhibition of Src kinase. E . CXCL1 expression by ELISA of HAdV-D37 infected or mock infected corneas at 16 hr post infection pretreated with the Src kinase inhibitor PP2 (10 μM) or DMSO control demonstrates a significant reduction in chemokine expression due to Src inhibition (*p

    Techniques Used: Mouse Assay, Injection, Labeling, Confocal Microscopy, Staining, Infection, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Inhibition

    Cholesterol depletion impedes viral entry. A . Human corneal cells grown on slide chambers were pretreated with MβCD (5 mM), RGD-containing 15-mer (50 μM), a KGE-containing 15-mer otherwise identical to the RGD 15-mer (50 μM), or the Src kinase inhibitor PP2 (10 μM), and then infected with Cy3-labeled HAdV-D37 (red). MβCD pretreatment prevented virus from entering the cells both at 30 min and 1 hr post infection as compared to no pretreatment. RGD and PP2 reduced entry as compared to KGE treated or untreated cells. Co-staining with alexa-fluor488 phalloidin (green). Original magnification: 63X. Insets represent similarly magnified squares from each photomicrograph. B . Cholesterol was added to MβCD pretreated cells for one hr and then infected with Cy3-labeled HAdV-D37. Confocal microscopy revealed entry of viruses into the corneal cells. C . Detergent free lipid raft preparations in HAdV-D37 infected corneal cells, pretreated with MβCD, RGD 15-mer, KGE 15-mer, or PP2, and lysed at 1 hr post infection, followed by immunoblotting for caveolin-1 or phosphorylated Src (pSrc). Blots show increased caveolin-1 and pSrc upon viral infection, but reduced expression with cholesterol depletion or blocking of integrin aggregation (MβCD and RGD treatment, respectively). PP2 treatment did not effect caveolin-1 levels in infected cells, but did reduce pSrc.
    Figure Legend Snippet: Cholesterol depletion impedes viral entry. A . Human corneal cells grown on slide chambers were pretreated with MβCD (5 mM), RGD-containing 15-mer (50 μM), a KGE-containing 15-mer otherwise identical to the RGD 15-mer (50 μM), or the Src kinase inhibitor PP2 (10 μM), and then infected with Cy3-labeled HAdV-D37 (red). MβCD pretreatment prevented virus from entering the cells both at 30 min and 1 hr post infection as compared to no pretreatment. RGD and PP2 reduced entry as compared to KGE treated or untreated cells. Co-staining with alexa-fluor488 phalloidin (green). Original magnification: 63X. Insets represent similarly magnified squares from each photomicrograph. B . Cholesterol was added to MβCD pretreated cells for one hr and then infected with Cy3-labeled HAdV-D37. Confocal microscopy revealed entry of viruses into the corneal cells. C . Detergent free lipid raft preparations in HAdV-D37 infected corneal cells, pretreated with MβCD, RGD 15-mer, KGE 15-mer, or PP2, and lysed at 1 hr post infection, followed by immunoblotting for caveolin-1 or phosphorylated Src (pSrc). Blots show increased caveolin-1 and pSrc upon viral infection, but reduced expression with cholesterol depletion or blocking of integrin aggregation (MβCD and RGD treatment, respectively). PP2 treatment did not effect caveolin-1 levels in infected cells, but did reduce pSrc.

    Techniques Used: Infection, Labeling, Staining, Confocal Microscopy, Expressing, Blocking Assay

    42) Product Images from "GW5074 and PP2 kinase inhibitors implicate nontraditional c-Raf and Lyn function as drivers of retinoic acid-induced maturation"

    Article Title: GW5074 and PP2 kinase inhibitors implicate nontraditional c-Raf and Lyn function as drivers of retinoic acid-induced maturation

    Journal: Cellular signalling

    doi: 10.1016/j.cellsig.2015.03.014

    Phenotypic markers during combined GW5074, PP2 and RA treatment. R38+ and R38− HL-60 cells were treated for 48 h with RA, or RA combined with GW5074 (GW) and/or PP2 and analyzed by flow cytometry for (A) CD38 and CD11b expressions and (B) G1/G0
    Figure Legend Snippet: Phenotypic markers during combined GW5074, PP2 and RA treatment. R38+ and R38− HL-60 cells were treated for 48 h with RA, or RA combined with GW5074 (GW) and/or PP2 and analyzed by flow cytometry for (A) CD38 and CD11b expressions and (B) G1/G0

    Techniques Used: Flow Cytometry, Cytometry

    Quantified signaling factor expression during combined GW5074, PP2 and RA treatment and correlation analysis. 48 h Western blot data (at least three repeats) of total lysates were quantified using ImageJ and fold change to respective R38+ or R38−
    Figure Legend Snippet: Quantified signaling factor expression during combined GW5074, PP2 and RA treatment and correlation analysis. 48 h Western blot data (at least three repeats) of total lysates were quantified using ImageJ and fold change to respective R38+ or R38−

    Techniques Used: Expressing, Western Blot

    43) Product Images from "Extracellular matrix-specific focal adhesions in vascular smooth muscle produce mechanically active adhesion sites"

    Article Title: Extracellular matrix-specific focal adhesions in vascular smooth muscle produce mechanically active adhesion sites

    Journal:

    doi: 10.1152/ajpcell.00516.2007

    A and B : VSMC mechanoresponses to FN-coated ( A ) and CNI-coated ( B ) beads in the presence of PP2 or PP3. VSMCs were incubated with PP2 (5 μM) or PP3 (5 μM) for 30 min before the application of AFM pulling force and measurement of bead displacement.
    Figure Legend Snippet: A and B : VSMC mechanoresponses to FN-coated ( A ) and CNI-coated ( B ) beads in the presence of PP2 or PP3. VSMCs were incubated with PP2 (5 μM) or PP3 (5 μM) for 30 min before the application of AFM pulling force and measurement of bead displacement.

    Techniques Used: Incubation

    44) Product Images from "Induction of TGF-?1 Synthesis by Macrophages in Response to Apoptotic Cells Requires Activation of the Scavenger Receptor CD36"

    Article Title: Induction of TGF-?1 Synthesis by Macrophages in Response to Apoptotic Cells Requires Activation of the Scavenger Receptor CD36

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0072772

    Both Lyn kinase and ERK1/2 MAPK are required for TGF-β1 synthesis induced by activating anti-CD36 mIgA. A, RAWTβRII cells were stimulated with activating anti-CD36 mIgA (2 µg/ml) for the times indicated. Total cell lysates were immunoblotted for phospho-Lyn kinase and the band density was normalized to total Lyn kinase. B, RAWTβRII cells were pretreated with the src-family kinase inhibitor PP2 (0.001 to 100 µM) for 2 h and then stimulated with anti-CD36 mIgA (2 µg/ml). After 6 h, TGF-β1 mRNA expression was analyzed by real-time PCR and normalized to GAPDH. Total TGF-β1 in the conditioned medium was analyzed by ELISA after 18 h. C, A time course of ERK1/2 phosphorylation was assessed by Western blotting in RAWTβRII cells treated with anti-CD36 mIgA (2 µg/ml). Phospho-ERK1/2 band density was normalized to total ERK1/2. D, RAWTβRII cells were preincubated with MEK kinase inhibitor U0126 or inactive analogue U0124 for 2 h and then stimulated with anti-CD36 mIgA for 6 h to detect TGF-β1 mRNA expression or for 18 h to detect secreted TGF-β1 protein as in Figure 1. Values represent means ± SD of six separate experiments.
    Figure Legend Snippet: Both Lyn kinase and ERK1/2 MAPK are required for TGF-β1 synthesis induced by activating anti-CD36 mIgA. A, RAWTβRII cells were stimulated with activating anti-CD36 mIgA (2 µg/ml) for the times indicated. Total cell lysates were immunoblotted for phospho-Lyn kinase and the band density was normalized to total Lyn kinase. B, RAWTβRII cells were pretreated with the src-family kinase inhibitor PP2 (0.001 to 100 µM) for 2 h and then stimulated with anti-CD36 mIgA (2 µg/ml). After 6 h, TGF-β1 mRNA expression was analyzed by real-time PCR and normalized to GAPDH. Total TGF-β1 in the conditioned medium was analyzed by ELISA after 18 h. C, A time course of ERK1/2 phosphorylation was assessed by Western blotting in RAWTβRII cells treated with anti-CD36 mIgA (2 µg/ml). Phospho-ERK1/2 band density was normalized to total ERK1/2. D, RAWTβRII cells were preincubated with MEK kinase inhibitor U0126 or inactive analogue U0124 for 2 h and then stimulated with anti-CD36 mIgA for 6 h to detect TGF-β1 mRNA expression or for 18 h to detect secreted TGF-β1 protein as in Figure 1. Values represent means ± SD of six separate experiments.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot

    Lyn kinase and ERK1/2 MAPK act by separate pathways for activating anti-CD36 mIgA-induced TGF-β1 synthesis. A, RAWTβRII cells were pretreated with inhibitor PP2 (30 µM) for 2 h prior to stimulation with anti-CD36 mIgA (2 µg/ml) for 60 min. Phosphorylation of Lyn and ERK1/2 were analyzed by Western blotting using total cell lysates. B, RAWTβRII cells were pretreated with inhibitor U0126 or analogue U0124 (1.0 µM) for 2 h and then incubated with anti-CD36 mIgA (2 µg/ml) for 90 min. Total cell lysates were used to analyze phosphorylation of Lyn and ERK1/2. Relative values for phosphorylated kinase versus total kinase were determined by densitometry and expressed as means ± SD of five separate experiments. *** P
    Figure Legend Snippet: Lyn kinase and ERK1/2 MAPK act by separate pathways for activating anti-CD36 mIgA-induced TGF-β1 synthesis. A, RAWTβRII cells were pretreated with inhibitor PP2 (30 µM) for 2 h prior to stimulation with anti-CD36 mIgA (2 µg/ml) for 60 min. Phosphorylation of Lyn and ERK1/2 were analyzed by Western blotting using total cell lysates. B, RAWTβRII cells were pretreated with inhibitor U0126 or analogue U0124 (1.0 µM) for 2 h and then incubated with anti-CD36 mIgA (2 µg/ml) for 90 min. Total cell lysates were used to analyze phosphorylation of Lyn and ERK1/2. Relative values for phosphorylated kinase versus total kinase were determined by densitometry and expressed as means ± SD of five separate experiments. *** P

    Techniques Used: Activated Clotting Time Assay, Western Blot, Incubation

    Lyn kinase and ERK1/2 are involved in CD36 mediated TGF-β1 induction by apoptotic cells. A, RAWTβRII cells were cultured in the presence of viable or apoptotic Jurkat cells for the time indicated. Total cell lysates were used for analyzing phosphorylation of ERK1/2 MAPK and Lyn kinase by Western blotting. B, RAWTβRII cells transfected with CD36-target siRNA or control siRNA (Ctr siRNA) for 24 h were incubated with apoptotic Jurkat cells for 60 or 90 min to analyze phosphorylation of Lyn kinase or ERK1/2, respectively. C, RAWTβRII cells were preincubated with PP2 (30 µM) or U0126 (1 µM) for 2h and then co-cultured with apoptotic Jurkat cells. TGF-β1 mRNA expression or secreted TGF-β1 protein was analyzed as in Figure 1. Values represent means ± SD of six separate experiments. ***, P
    Figure Legend Snippet: Lyn kinase and ERK1/2 are involved in CD36 mediated TGF-β1 induction by apoptotic cells. A, RAWTβRII cells were cultured in the presence of viable or apoptotic Jurkat cells for the time indicated. Total cell lysates were used for analyzing phosphorylation of ERK1/2 MAPK and Lyn kinase by Western blotting. B, RAWTβRII cells transfected with CD36-target siRNA or control siRNA (Ctr siRNA) for 24 h were incubated with apoptotic Jurkat cells for 60 or 90 min to analyze phosphorylation of Lyn kinase or ERK1/2, respectively. C, RAWTβRII cells were preincubated with PP2 (30 µM) or U0126 (1 µM) for 2h and then co-cultured with apoptotic Jurkat cells. TGF-β1 mRNA expression or secreted TGF-β1 protein was analyzed as in Figure 1. Values represent means ± SD of six separate experiments. ***, P

    Techniques Used: Cell Culture, Western Blot, Transfection, Incubation, Expressing

    45) Product Images from "Inactivation of TGF? signaling in neural crest stem cells leads to multiple defects reminiscent of DiGeorge syndrome"

    Article Title: Inactivation of TGF? signaling in neural crest stem cells leads to multiple defects reminiscent of DiGeorge syndrome

    Journal: Genes & Development

    doi: 10.1101/gad.317405

    TGFβ-dependent CrkL phosphorylation and neural crest differentiation. ( A ) Mutant (mt) neural crest cells expressing βGal (blue) in branchial arch 1 (ba1), branchial arch 2 (Supplementary Fig. 1), and the developing aorto-pulmonary septum (arrowheads) at E10 show strongly reduced phosphorylation of CrkL (pCrkL; brown; gross and detailed view) and lack nuclear phospho-Smad2 (brown; detailed view) compared with control (co). Note comparable staining intensity for phosphorylated CrkL in βGal-negative tissue (arrow) in mutant (mt) and control (co). ( B ) Immortalized NCSCs (Monc1 cells) express smooth muscle α-actin (SMαA) upon treatment with TGFβ, while untreated (n.a.) cells are SMαA-negative. ( C ) Western blot analysis reveals increased Smad2, CrkL, and Src phosphorylation by TGFβ in Monc1 cells. ( D ) CrkL phosphorylation in TGFβ-treated Monc1 cells is reduced to levels of untreated cells in the presence of Src kinase inhibitors PP1 and PP2, respectively. ( E ) βGal-expressing neural crest cells populate the pharyngeal apparatus (saggital section). (ba1-3) Branchial arches 1-3; (bp3/4) branchial pouches 3/4; (baa3/4) branchial arch arteries 3/4; (paps) prospective aorto-pulmonary septum. ( F ) Neural crest cells (NC; blue) present in the pharyngeal apparatus require TGFβ-signaling for CrkL phosphorylation and for expression of the non-neural markers sox9 in the first branchial arch and SMαA in the forming septum of the heart outflow tract. ( G ) Similar to neural crest cells in Crkol -mutant mice, T β RII -deficient neural crest cells migrate normally into the pharyngeal apparatus. Here, the signal adaptor protein CrkL fails to be phosphorylated in response to TGFβ, and mutant neural crest cells fail to express sox9 and SMαA. Tbx1 expression in the branchial pouch endoderm and early pharyngeal arch patterning is not affected. The failure of mutant neural crest cells to acquire non-neural fates in the early pharyngeal apparatus impairs the development of tissues derived from the pharyngeal apparatus and leads to a DiGeorge-like phenotype.
    Figure Legend Snippet: TGFβ-dependent CrkL phosphorylation and neural crest differentiation. ( A ) Mutant (mt) neural crest cells expressing βGal (blue) in branchial arch 1 (ba1), branchial arch 2 (Supplementary Fig. 1), and the developing aorto-pulmonary septum (arrowheads) at E10 show strongly reduced phosphorylation of CrkL (pCrkL; brown; gross and detailed view) and lack nuclear phospho-Smad2 (brown; detailed view) compared with control (co). Note comparable staining intensity for phosphorylated CrkL in βGal-negative tissue (arrow) in mutant (mt) and control (co). ( B ) Immortalized NCSCs (Monc1 cells) express smooth muscle α-actin (SMαA) upon treatment with TGFβ, while untreated (n.a.) cells are SMαA-negative. ( C ) Western blot analysis reveals increased Smad2, CrkL, and Src phosphorylation by TGFβ in Monc1 cells. ( D ) CrkL phosphorylation in TGFβ-treated Monc1 cells is reduced to levels of untreated cells in the presence of Src kinase inhibitors PP1 and PP2, respectively. ( E ) βGal-expressing neural crest cells populate the pharyngeal apparatus (saggital section). (ba1-3) Branchial arches 1-3; (bp3/4) branchial pouches 3/4; (baa3/4) branchial arch arteries 3/4; (paps) prospective aorto-pulmonary septum. ( F ) Neural crest cells (NC; blue) present in the pharyngeal apparatus require TGFβ-signaling for CrkL phosphorylation and for expression of the non-neural markers sox9 in the first branchial arch and SMαA in the forming septum of the heart outflow tract. ( G ) Similar to neural crest cells in Crkol -mutant mice, T β RII -deficient neural crest cells migrate normally into the pharyngeal apparatus. Here, the signal adaptor protein CrkL fails to be phosphorylated in response to TGFβ, and mutant neural crest cells fail to express sox9 and SMαA. Tbx1 expression in the branchial pouch endoderm and early pharyngeal arch patterning is not affected. The failure of mutant neural crest cells to acquire non-neural fates in the early pharyngeal apparatus impairs the development of tissues derived from the pharyngeal apparatus and leads to a DiGeorge-like phenotype.

    Techniques Used: Mutagenesis, Expressing, Staining, Western Blot, Papanicolaou Stain, Mouse Assay, Derivative Assay

    46) Product Images from "N-WASP-directed actin polymerization activates Cas phosphorylation and lamellipodium spreading"

    Article Title: N-WASP-directed actin polymerization activates Cas phosphorylation and lamellipodium spreading

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.134692

    SFKs downstream of integrins phosphorylate CasSD in an anchorage-dependent manner. (A–D) Inhibition of SFK by use of PP2 diminished fibronectin-induced CasSD phosphorylation at residues Y165 and Y410 but caused hyperphosphorylation of SFK at residue Y416, as shown by western blot (IB) (A). sus, suspension. Cells were pre-incubated in 20 µM PP2 or its control chemical PP3 for 30 min before being plated on fibronectin-coated dishes (FN). The ratios of (B) pY165 CasSD∶total Cas and (C) pY410 CasSD∶total Cas in different experiments were normalized against control samples at the 30-min time-point and (D) pY416 Src∶total Src ratios against control samples at the 10-min time-point; mean±s.d., four repeats. P -values were calculated by two-tailed paired Student's t -test. A.U., arbitrary units. (E,F) Immunostaining of (E) PP3-treated or (F) PP2-treated early spreading cells. Cells treated with PP2 showed a delay in spreading and defective formation of active protrusions. Sites of limited Cas phosphorylation signal correlated with local actin polymerization (F). Cells were fixed 10 min after spreading initiation. Scale bar: 10 µm. (G) Interaction of β3 integrins with Src was detected in cells in suspension and adherent cells. Fibroblasts were transfected with a β3-integrin–GFP plasmid and harvested 24 h later, kept in suspension for 45 min and then directly lysed with RIPA buffer or plated onto a fibronectin-coated dish for 15 min before lysis. 2 µl of polyclonal antibody was used to pull down endogenous and overexpressed β3 integrins. The mock sample was prepared from the lysate of cells in suspension and no antibody was applied. Src associated with the immunoprecipitated (IP) β3 integrins was detected using an antibody against Src (shown in H). The level of β3 integrin pulled down was monitored using an antibody against GFP. (H–J) The SFK activity of suspension cells increased in response to RGD soluble peptide. Application of RGD did not affect the SFK activity in early spreading cells although CasSD phosphorylation showed a significant decrease. * P
    Figure Legend Snippet: SFKs downstream of integrins phosphorylate CasSD in an anchorage-dependent manner. (A–D) Inhibition of SFK by use of PP2 diminished fibronectin-induced CasSD phosphorylation at residues Y165 and Y410 but caused hyperphosphorylation of SFK at residue Y416, as shown by western blot (IB) (A). sus, suspension. Cells were pre-incubated in 20 µM PP2 or its control chemical PP3 for 30 min before being plated on fibronectin-coated dishes (FN). The ratios of (B) pY165 CasSD∶total Cas and (C) pY410 CasSD∶total Cas in different experiments were normalized against control samples at the 30-min time-point and (D) pY416 Src∶total Src ratios against control samples at the 10-min time-point; mean±s.d., four repeats. P -values were calculated by two-tailed paired Student's t -test. A.U., arbitrary units. (E,F) Immunostaining of (E) PP3-treated or (F) PP2-treated early spreading cells. Cells treated with PP2 showed a delay in spreading and defective formation of active protrusions. Sites of limited Cas phosphorylation signal correlated with local actin polymerization (F). Cells were fixed 10 min after spreading initiation. Scale bar: 10 µm. (G) Interaction of β3 integrins with Src was detected in cells in suspension and adherent cells. Fibroblasts were transfected with a β3-integrin–GFP plasmid and harvested 24 h later, kept in suspension for 45 min and then directly lysed with RIPA buffer or plated onto a fibronectin-coated dish for 15 min before lysis. 2 µl of polyclonal antibody was used to pull down endogenous and overexpressed β3 integrins. The mock sample was prepared from the lysate of cells in suspension and no antibody was applied. Src associated with the immunoprecipitated (IP) β3 integrins was detected using an antibody against Src (shown in H). The level of β3 integrin pulled down was monitored using an antibody against GFP. (H–J) The SFK activity of suspension cells increased in response to RGD soluble peptide. Application of RGD did not affect the SFK activity in early spreading cells although CasSD phosphorylation showed a significant decrease. * P

    Techniques Used: Inhibition, Western Blot, Incubation, Two Tailed Test, Immunostaining, Transfection, Plasmid Preparation, Lysis, Immunoprecipitation, Activity Assay

    47) Product Images from "Progesterone Receptors Upregulate Wnt-1 To Induce Epidermal Growth Factor Receptor Transactivation and c-Src-Dependent Sustained Activation of Erk1/2 Mitogen-Activated Protein Kinase in Breast Cancer Cells ▿"

    Article Title: Progesterone Receptors Upregulate Wnt-1 To Induce Epidermal Growth Factor Receptor Transactivation and c-Src-Dependent Sustained Activation of Erk1/2 Mitogen-Activated Protein Kinase in Breast Cancer Cells ▿

    Journal:

    doi: 10.1128/MCB.01539-06

    Persistent c-Src activation is required for progestin-induced sustained Erk1/2 activity. (A) T47D-YB cells were pretreated for 30 min with DMSO vehicle control, U0126, the c-Src family inhibitor PP2 or SU6656 (SU), or the PI3-K inhibitor, LY294002 (LY),
    Figure Legend Snippet: Persistent c-Src activation is required for progestin-induced sustained Erk1/2 activity. (A) T47D-YB cells were pretreated for 30 min with DMSO vehicle control, U0126, the c-Src family inhibitor PP2 or SU6656 (SU), or the PI3-K inhibitor, LY294002 (LY),

    Techniques Used: Activation Assay, Activity Assay

    48) Product Images from "Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan"

    Article Title: Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan

    Journal: Cellular microbiology

    doi: 10.1111/cmi.12078

    Binding of LASV to cellular DG induces tyrosine phosphorylation of β-DG by src-family kinases (A) Schematic representation of C-terminally tagged DG (DGHA). The N-terminal domain (white), the mucin-type domain (black) and the C-terminal domain (gray) of α-DG, β-DG, and the C-terminal HA tag are indicated. (B). Detection of tyrosine phosphorylation at residue Y892 with mAb cl14a. DGHA was transiently expressed either alone or in combination with c-src. Parallel specimens were pretreated with 20 μM PP2 or mock treated with vehicle (DMSO). After 48 hours, DGHA was isolated by pull-down with HA matrix. Proteins were separated and probed in Western blot with an antibody to HA (anti-HA) or mAb cl14a to β-DG phosphorylated at tyrosine 892 (anti-β-DG PY892). Apparent molecular masses and the positions of β-DG are indicated. (C) Attachment of rLCMVLASVGP to cells induces tyrosine phosphorylation of β-DG. Monolayers of WI-26 VA4 cells were incubated with rLCMV-LASVGP or PICV (100 particles/cell) for 1 hour in the cold. Unbound virus was removed and cells shifted to 37°C. At the indicated time points, cells were lysed and DG enriched by WGA affinity purification. WGA-bound glycoproteins were probed in Western-blot with mAb cl14a (anti-β-DG PY892) and antibody 8D5 to β-DG. The positions of β-DG and β-DG PY892 are indicated. (D) Virus induced tyrosine phosphorylation of β-DG is blocked by PP2. Monolayers of WI-26 VA4 cells were pretreated with 20 μM PP2 or DMSO (Control) for 1 hour prior to exposure to rLCMV-LASVGP. Virus-induced phosphorylation of β-DG at Y892 was assessed as in (C). (E) The phosphorylation of β-DG at PY892 is not required for LASV cell entry. Monolayers of WI-26 VA4 cells were pretreated with 20 μM PP2 or DMSO (Control) for 1 hour as in (D), followed by incubation with rLCMV-LASVGP (MOI = 1) in the cold in presence of the drug. After 1 hour, unbound virus was removed and pre-warmed (37 °C) medium containing the drug added. At the indicated time points, 20 mM ammonium chloride was added and left throughout the experiment. At 16 hours post infection, cells were fixed and infection detected by intracellular staining for LCMV NP (means ± SD, n = 3). The apparent differences in infection at 60 minutes were not statistically significant.
    Figure Legend Snippet: Binding of LASV to cellular DG induces tyrosine phosphorylation of β-DG by src-family kinases (A) Schematic representation of C-terminally tagged DG (DGHA). The N-terminal domain (white), the mucin-type domain (black) and the C-terminal domain (gray) of α-DG, β-DG, and the C-terminal HA tag are indicated. (B). Detection of tyrosine phosphorylation at residue Y892 with mAb cl14a. DGHA was transiently expressed either alone or in combination with c-src. Parallel specimens were pretreated with 20 μM PP2 or mock treated with vehicle (DMSO). After 48 hours, DGHA was isolated by pull-down with HA matrix. Proteins were separated and probed in Western blot with an antibody to HA (anti-HA) or mAb cl14a to β-DG phosphorylated at tyrosine 892 (anti-β-DG PY892). Apparent molecular masses and the positions of β-DG are indicated. (C) Attachment of rLCMVLASVGP to cells induces tyrosine phosphorylation of β-DG. Monolayers of WI-26 VA4 cells were incubated with rLCMV-LASVGP or PICV (100 particles/cell) for 1 hour in the cold. Unbound virus was removed and cells shifted to 37°C. At the indicated time points, cells were lysed and DG enriched by WGA affinity purification. WGA-bound glycoproteins were probed in Western-blot with mAb cl14a (anti-β-DG PY892) and antibody 8D5 to β-DG. The positions of β-DG and β-DG PY892 are indicated. (D) Virus induced tyrosine phosphorylation of β-DG is blocked by PP2. Monolayers of WI-26 VA4 cells were pretreated with 20 μM PP2 or DMSO (Control) for 1 hour prior to exposure to rLCMV-LASVGP. Virus-induced phosphorylation of β-DG at Y892 was assessed as in (C). (E) The phosphorylation of β-DG at PY892 is not required for LASV cell entry. Monolayers of WI-26 VA4 cells were pretreated with 20 μM PP2 or DMSO (Control) for 1 hour as in (D), followed by incubation with rLCMV-LASVGP (MOI = 1) in the cold in presence of the drug. After 1 hour, unbound virus was removed and pre-warmed (37 °C) medium containing the drug added. At the indicated time points, 20 mM ammonium chloride was added and left throughout the experiment. At 16 hours post infection, cells were fixed and infection detected by intracellular staining for LCMV NP (means ± SD, n = 3). The apparent differences in infection at 60 minutes were not statistically significant.

    Techniques Used: Binding Assay, Isolation, Western Blot, Incubation, Whole Genome Amplification, Affinity Purification, Infection, Staining

    Effect of LASV pseudotype binding on the association of DG with utrophin Monolayers of WI-26 VA4 cells were chilled on ice and incubated with either LASV or AMPV pseudotypes (LASV-PS, AMPV-PS) at a multiplicity of infection (MOI) of 50 transforming units (TU)/cell. Parallel specimens were incubated with mAb 16G4 to α-DG (anti-DG). After one hour, unbound virus or mAb were removed by washing. Cells were either kept on ice (virus binding 4°C) or shifted to 37 °C for 10 minutes (temperature shift 37°C). Cells were quickly chilled on ice, lysed and subjected to IP using FLAG matrix or protein G-conjugated Sepharose 4B. Immunocomplexes were separated by SDS-PAGE using 100% of the IPs anti-FLAG and 5% of the IP anti-DG. Beta-DG and utrophin were detected on Western-blot using monoclonal antibodies 8D5 and combined with HRP-conjugated secondary antibodies in a TrueBlot® detection system to avoid cross-reaction with the IgG heavy chain. For the detection of total protein in cell lysates, 1/20 of the lysate were separated by SDS-PAGE and subjected to Western-blot detection. (B) Quantification of the signals in (A). Blots were scanned in a densitometer and the ratios of the signals for utrophin normalized to β-DG (utrophin/β-DG) for the IPs anti-FLAG (LASV pseudotypes only) and the IP anti-DG. For each series, the utrophin/β-DG ratio detected in the IP anti-DG was defined as 1.0. (C) Pre-treatment with genistein, but not PP2 reduced virus-induced dissociation of utrophin from DG. Monolayers of WI-26 VA4 cells were pre-treated with DMSO only (control), 20 μM PP2 and 50 μM genistein for one hour. Cells were then chilled on ice and incubated with LASV pseudotypes (LASV-PS) for one hour in the cold in presence of drugs. Cells were then quickly shifted to 37 °C, lysed, and subjected to IP with FLAG matrix as in (A). Precipitated β-DG and utrophin were detected in Western blot and the ratios utrophin/β-DG determined as in (B). (D) Quantification of the data in (C). (E) The 15 C-terminal amino acids of β-DG are dispensable for LASV cell entry. Murine ES cells expressing either wild-type DG (DG wt) or DG lacking the C-terminal 15 amino acids of β-DG (DGΔC) were infected with rLCMV-LASVGP or rLCMV-VSVG at a multiplicity of 0.1. Infection of the cells expressing wild-type DG was set at 100% (means ± SD, n = 3).
    Figure Legend Snippet: Effect of LASV pseudotype binding on the association of DG with utrophin Monolayers of WI-26 VA4 cells were chilled on ice and incubated with either LASV or AMPV pseudotypes (LASV-PS, AMPV-PS) at a multiplicity of infection (MOI) of 50 transforming units (TU)/cell. Parallel specimens were incubated with mAb 16G4 to α-DG (anti-DG). After one hour, unbound virus or mAb were removed by washing. Cells were either kept on ice (virus binding 4°C) or shifted to 37 °C for 10 minutes (temperature shift 37°C). Cells were quickly chilled on ice, lysed and subjected to IP using FLAG matrix or protein G-conjugated Sepharose 4B. Immunocomplexes were separated by SDS-PAGE using 100% of the IPs anti-FLAG and 5% of the IP anti-DG. Beta-DG and utrophin were detected on Western-blot using monoclonal antibodies 8D5 and combined with HRP-conjugated secondary antibodies in a TrueBlot® detection system to avoid cross-reaction with the IgG heavy chain. For the detection of total protein in cell lysates, 1/20 of the lysate were separated by SDS-PAGE and subjected to Western-blot detection. (B) Quantification of the signals in (A). Blots were scanned in a densitometer and the ratios of the signals for utrophin normalized to β-DG (utrophin/β-DG) for the IPs anti-FLAG (LASV pseudotypes only) and the IP anti-DG. For each series, the utrophin/β-DG ratio detected in the IP anti-DG was defined as 1.0. (C) Pre-treatment with genistein, but not PP2 reduced virus-induced dissociation of utrophin from DG. Monolayers of WI-26 VA4 cells were pre-treated with DMSO only (control), 20 μM PP2 and 50 μM genistein for one hour. Cells were then chilled on ice and incubated with LASV pseudotypes (LASV-PS) for one hour in the cold in presence of drugs. Cells were then quickly shifted to 37 °C, lysed, and subjected to IP with FLAG matrix as in (A). Precipitated β-DG and utrophin were detected in Western blot and the ratios utrophin/β-DG determined as in (B). (D) Quantification of the data in (C). (E) The 15 C-terminal amino acids of β-DG are dispensable for LASV cell entry. Murine ES cells expressing either wild-type DG (DG wt) or DG lacking the C-terminal 15 amino acids of β-DG (DGΔC) were infected with rLCMV-LASVGP or rLCMV-VSVG at a multiplicity of 0.1. Infection of the cells expressing wild-type DG was set at 100% (means ± SD, n = 3).

    Techniques Used: Binding Assay, Incubation, Infection, SDS Page, Western Blot, Expressing

    49) Product Images from "A RhoG-mediated signaling pathway that modulates invadopodia dynamics in breast cancer cells"

    Article Title: A RhoG-mediated signaling pathway that modulates invadopodia dynamics in breast cancer cells

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.195552

    RhoG knockdown decreases paxillin phosphorylation at invadopodia. (A) Cell lysates from CTRL, RhoG KD and SGEF KD SUM159 cells were analyzed by western blotting and probed for phospho-paxillin (PXN p118), total paxillin (PXN) and tubulin as a loading control (left panel). The quantification represents the average of at least three independent experiments. Data are mean±s.e.m. (right panel). (B) CTRL and RhoG KD SUM159 cells were treated with PDBu for 30 min, fixed and stained with Alexa-Fluor-647–phalloidin (magenta), and for PXN (green) and PXN p118 (red). Scale bars: 10 µm. (C) Graphs indicate fluorescent intensity in arbitrary units (A.U.) of PXN p118 (red) with respect to PXN (green) and F-actin (blue) over the indicated line scan in (B). (D) Total fluorescence intensity for PXN and PXN p118 in individual invadopodia from CTRL and RhoG KD cells were measurements of at least 50 invadopodia per condition. Data are mean±s.e.m. (E) CTRL and RhoG KD cells were cultured in the presence of vehicle control, 5 μM PP2 or 5 μM PF-573228 for 30 min. Cell lysates were analyzed by western blotting and probed for total paxillin (PXN) and phosho-paxillin (PXN p118). (F) Quantification of PXN p118 to PXN ratio in cells treated with vehicle, PP2 and PF-573228. Results represent the mean±s.e.m. of at least three independent experiments. * P
    Figure Legend Snippet: RhoG knockdown decreases paxillin phosphorylation at invadopodia. (A) Cell lysates from CTRL, RhoG KD and SGEF KD SUM159 cells were analyzed by western blotting and probed for phospho-paxillin (PXN p118), total paxillin (PXN) and tubulin as a loading control (left panel). The quantification represents the average of at least three independent experiments. Data are mean±s.e.m. (right panel). (B) CTRL and RhoG KD SUM159 cells were treated with PDBu for 30 min, fixed and stained with Alexa-Fluor-647–phalloidin (magenta), and for PXN (green) and PXN p118 (red). Scale bars: 10 µm. (C) Graphs indicate fluorescent intensity in arbitrary units (A.U.) of PXN p118 (red) with respect to PXN (green) and F-actin (blue) over the indicated line scan in (B). (D) Total fluorescence intensity for PXN and PXN p118 in individual invadopodia from CTRL and RhoG KD cells were measurements of at least 50 invadopodia per condition. Data are mean±s.e.m. (E) CTRL and RhoG KD cells were cultured in the presence of vehicle control, 5 μM PP2 or 5 μM PF-573228 for 30 min. Cell lysates were analyzed by western blotting and probed for total paxillin (PXN) and phosho-paxillin (PXN p118). (F) Quantification of PXN p118 to PXN ratio in cells treated with vehicle, PP2 and PF-573228. Results represent the mean±s.e.m. of at least three independent experiments. * P

    Techniques Used: Western Blot, Staining, Fluorescence, Cell Culture

    Src activity is necessary for increased invadopodia formation after RhoG KD. Western blot analysis shows increased levels of phospho-Src (Src p418) (A) and phospho-FAK (FAK p397) (B) in RhoG-depleted cells. Data are mean±s.e.m. of three independent experiments. (C–F) CTRL and RhoG KD cells were cultured in the presence of vehicle control, 5 μM PP2 (C,D) or 5 μM PF-573228 (E,F) for 30 min followed by a 30 min incubation with PDBu. The efficiency of the inhibitors was tested by immunoblotting for phospho-Src (Src p418) (C) or phospho-FAK (FAK p397) (E). Quantifications in D and F show the percentage of cells with invadopodia, as determined by cortactin and actin staining. Data are mean±s.e.m. of three independent experiments. * P
    Figure Legend Snippet: Src activity is necessary for increased invadopodia formation after RhoG KD. Western blot analysis shows increased levels of phospho-Src (Src p418) (A) and phospho-FAK (FAK p397) (B) in RhoG-depleted cells. Data are mean±s.e.m. of three independent experiments. (C–F) CTRL and RhoG KD cells were cultured in the presence of vehicle control, 5 μM PP2 (C,D) or 5 μM PF-573228 (E,F) for 30 min followed by a 30 min incubation with PDBu. The efficiency of the inhibitors was tested by immunoblotting for phospho-Src (Src p418) (C) or phospho-FAK (FAK p397) (E). Quantifications in D and F show the percentage of cells with invadopodia, as determined by cortactin and actin staining. Data are mean±s.e.m. of three independent experiments. * P

    Techniques Used: Activity Assay, Western Blot, Cell Culture, Incubation, Staining

    50) Product Images from "HGF-independent Potentiation of EGFR Action by c-Met"

    Article Title: HGF-independent Potentiation of EGFR Action by c-Met

    Journal: Oncogene

    doi: 10.1038/onc.2011.84

    C-Src mediates EGFR-induced c-Met phosphorylation. 201T cells were serum deprived for 2 days followed by addition of EGF (10 nM) for 0–48 h. (A) Cell lysates were immunoprecipitated for total c-Src and analyzed for phospho-c-Src (Y418). (B) 201T cells were pretreated for 2 h with SFK inhibitors PP2 (500 nM) or dasatinib (50 nM) prior to 24 h EGF (10 nM) or 5 min HGF (10 ng/mL) stimulation, or were stably transfected with either a dominant-negative c-Src construct (DN-Src) or empty vector (EV) and stimulated with EGF. Cell lysates were prepared and analyzed for phospho- and total c-Met levels. (C) A549 and 201T cells were pretreated for 2 h with PP2 (500 nM) prior to 24 h EGF stimulation, mRNA was harvested, and subjected to c-Met quantitative RT-PCR using β-Gus as an internal control.
    Figure Legend Snippet: C-Src mediates EGFR-induced c-Met phosphorylation. 201T cells were serum deprived for 2 days followed by addition of EGF (10 nM) for 0–48 h. (A) Cell lysates were immunoprecipitated for total c-Src and analyzed for phospho-c-Src (Y418). (B) 201T cells were pretreated for 2 h with SFK inhibitors PP2 (500 nM) or dasatinib (50 nM) prior to 24 h EGF (10 nM) or 5 min HGF (10 ng/mL) stimulation, or were stably transfected with either a dominant-negative c-Src construct (DN-Src) or empty vector (EV) and stimulated with EGF. Cell lysates were prepared and analyzed for phospho- and total c-Met levels. (C) A549 and 201T cells were pretreated for 2 h with PP2 (500 nM) prior to 24 h EGF stimulation, mRNA was harvested, and subjected to c-Met quantitative RT-PCR using β-Gus as an internal control.

    Techniques Used: Immunoprecipitation, Stable Transfection, Transfection, Dominant Negative Mutation, Construct, Plasmid Preparation, Quantitative RT-PCR

    51) Product Images from "Proto-Oncogenic Src Phosphorylates EB1 to Regulate the Microtubule-Focal Adhesion Crosstalk and Stimulate Cell Migration"

    Article Title: Proto-Oncogenic Src Phosphorylates EB1 to Regulate the Microtubule-Focal Adhesion Crosstalk and Stimulate Cell Migration

    Journal: Theranostics

    doi: 10.7150/thno.16356

    EB1 phosphorylation at Y247 regulates MT dynamics. (A) MT growth tracks were overlaid and classified into four groups and color-coded based on the average MT growth velocity and lifetime. (B) HUVECs were transfected with GFP-EB1 wild-type, Y247F, or Y247D, and time-lapse videos of GFP-EB1 comets were taken at 2-s intervals. MT growth tracks were then analyzed with the plusTipTracker software. Scale bars, 10 μm. (C) Experiments were performed as in (B), and MT growth velocity and lifetime were measured (20-30 cells were measured for each group). (D) HUVECs were transfected with GFP-EB1 and treated with PP2 (10 μM) or equal amount of DMSO for 6 h. MT growth tracks were then analyzed as in (B). Scale bars, 10 μm. (E) Experiments were performed as in (D), and MT growth velocity and lifetime were then measured (20-30 cells were measured for each group). (F) HUVECs were transfected with GFP-EB1 and HA-Src or the HA vector. MT growth tracks were then analyzed as in (B). Scale bars, 10 μm. (G) Experiments were performed as in (F), and MT growth velocity and lifetime were then measured (20-30 cells were measured for each group). (H) HUVECs were transfected with siControl or siSrc, and immunoblotting was then performed with the indicated antibodies. (I) HUVECs were transfected with GFP-EB1 and siControl or siSrc. MT growth tracks were then analyzed as in (B). Scale bars, 10 μm. (J) Experiments were performed as in (I), and MT growth velocity and lifetime were then measured (20-30 cells were measured for each group). All experiments were replicated three times. * p
    Figure Legend Snippet: EB1 phosphorylation at Y247 regulates MT dynamics. (A) MT growth tracks were overlaid and classified into four groups and color-coded based on the average MT growth velocity and lifetime. (B) HUVECs were transfected with GFP-EB1 wild-type, Y247F, or Y247D, and time-lapse videos of GFP-EB1 comets were taken at 2-s intervals. MT growth tracks were then analyzed with the plusTipTracker software. Scale bars, 10 μm. (C) Experiments were performed as in (B), and MT growth velocity and lifetime were measured (20-30 cells were measured for each group). (D) HUVECs were transfected with GFP-EB1 and treated with PP2 (10 μM) or equal amount of DMSO for 6 h. MT growth tracks were then analyzed as in (B). Scale bars, 10 μm. (E) Experiments were performed as in (D), and MT growth velocity and lifetime were then measured (20-30 cells were measured for each group). (F) HUVECs were transfected with GFP-EB1 and HA-Src or the HA vector. MT growth tracks were then analyzed as in (B). Scale bars, 10 μm. (G) Experiments were performed as in (F), and MT growth velocity and lifetime were then measured (20-30 cells were measured for each group). (H) HUVECs were transfected with siControl or siSrc, and immunoblotting was then performed with the indicated antibodies. (I) HUVECs were transfected with GFP-EB1 and siControl or siSrc. MT growth tracks were then analyzed as in (B). Scale bars, 10 μm. (J) Experiments were performed as in (I), and MT growth velocity and lifetime were then measured (20-30 cells were measured for each group). All experiments were replicated three times. * p

    Techniques Used: Transfection, Software, Plasmid Preparation

    Src-mediated EB1 phosphorylation diminishes its interactions with APC-C and MCAK. (A) HEK293T cells were transfected with GFP-EB1 and HA-EB1 wild-type or Y247D. Immunoprecipitation and immunoblotting were then performed with the indicated antibodies. (B) HEK293T cells were transfected with GFP-APC-C and HA-EB1 wild-type, Y247F, or Y247D, or the HA vector. Immunoprecipitation and immunoblotting were then performed as indicated. (C) Experiments were performed as in (B) except that GFP-MCAK was used instead of GFP-APC-C. (D) Structural analysis of the interaction between the SxIP motif (violet) and EB1 wild-type, Y247F, or Y247D. (E) HEK293T cells were transfected with GFP-APC-C and treated with SU6656 (10 μM), PP2 (10 μM), or equal amount of DMSO for 12 h or co-transfected with HA-Src or the HA vector. Immunoprecipitation and immunoblotting were then performed. The arrowhead indicates the bands of GFP-APC-C. (F) Experiments were performed as in (D) except that GFP-MCAK was used instead of GFP-APC-C. The arrowhead indicates the bands of GFP-MCAK. All experiments were replicated three times.
    Figure Legend Snippet: Src-mediated EB1 phosphorylation diminishes its interactions with APC-C and MCAK. (A) HEK293T cells were transfected with GFP-EB1 and HA-EB1 wild-type or Y247D. Immunoprecipitation and immunoblotting were then performed with the indicated antibodies. (B) HEK293T cells were transfected with GFP-APC-C and HA-EB1 wild-type, Y247F, or Y247D, or the HA vector. Immunoprecipitation and immunoblotting were then performed as indicated. (C) Experiments were performed as in (B) except that GFP-MCAK was used instead of GFP-APC-C. (D) Structural analysis of the interaction between the SxIP motif (violet) and EB1 wild-type, Y247F, or Y247D. (E) HEK293T cells were transfected with GFP-APC-C and treated with SU6656 (10 μM), PP2 (10 μM), or equal amount of DMSO for 12 h or co-transfected with HA-Src or the HA vector. Immunoprecipitation and immunoblotting were then performed. The arrowhead indicates the bands of GFP-APC-C. (F) Experiments were performed as in (D) except that GFP-MCAK was used instead of GFP-APC-C. The arrowhead indicates the bands of GFP-MCAK. All experiments were replicated three times.

    Techniques Used: Transfection, Immunoprecipitation, Plasmid Preparation

    Src-mediated phosphorylation of EB1 enhances cell migration. (A) HUVECs were transfected with GFP-EB1 wild-type, Y247F, or Y247D, and cell migration trajectories were then recorded. Scale bars, 40 μm. (B and C) Experiments were performed as in (A), and the velocity of cell migration (B) and the distance from the origin (C) were measured (20-30 cells were measured for each group). (D) HUVECs were transfected with GFP-EB1 wild-type or Y247D and treated with PP2 or DMSO. Cell migration trajectories were then obtained. Scale bars, 40 μm. (E and F) Experiments were performed as in (D), and the velocity of cell migration (E) and the distance from the origin (F) were measured (20-30 cells were measured for each group). (G) HUVECs were transfected with siControl or siSrc, together with GFP-EB1 wild-type or Y247D. Cell migration trajectories were then obtained. Scale bars, 40 μm. (H and I) Experiments were performed as in (G), and the velocity of cell migration (H) and the distance from the origin (I) were measured (20-30 cells were measured for each group). All experiments were replicated three times. ** p
    Figure Legend Snippet: Src-mediated phosphorylation of EB1 enhances cell migration. (A) HUVECs were transfected with GFP-EB1 wild-type, Y247F, or Y247D, and cell migration trajectories were then recorded. Scale bars, 40 μm. (B and C) Experiments were performed as in (A), and the velocity of cell migration (B) and the distance from the origin (C) were measured (20-30 cells were measured for each group). (D) HUVECs were transfected with GFP-EB1 wild-type or Y247D and treated with PP2 or DMSO. Cell migration trajectories were then obtained. Scale bars, 40 μm. (E and F) Experiments were performed as in (D), and the velocity of cell migration (E) and the distance from the origin (F) were measured (20-30 cells were measured for each group). (G) HUVECs were transfected with siControl or siSrc, together with GFP-EB1 wild-type or Y247D. Cell migration trajectories were then obtained. Scale bars, 40 μm. (H and I) Experiments were performed as in (G), and the velocity of cell migration (H) and the distance from the origin (I) were measured (20-30 cells were measured for each group). All experiments were replicated three times. ** p

    Techniques Used: Migration, Transfection

    Src interacts with and phosphorylates EB1 both in cells and in vitro . (A) HEK293T cells were transfected with HA-EB1 and GFP-Src or the GFP vector. Immunoprecipitation (IP) and immunoblotting (IB) were then performed with the indicated antibodies. (B) HEK293T cells were treated with SU6656 (10 μM), PP2 (10 μM), or equal amount of the vehicle DMSO for 12 h. Immunoprecipitation and immunoblotting were then performed as indicated. (C) In vitro kinase assays were performed with purified His-EB1 and the GFP or GFP-Src immunoprecipitate from HEK293T cells, and examined by immunoblotting. (D) In vitro kinase assays were performed with purified His-EB1 and the GFP-Src immunoprecipitate, in the absence (Mock) or presence of SU6656 (50 μM), PP2 (50 μM), or equal amount of DMSO, and analyzed by immunoblotting. (E) In vitro kinase assays were performed with purified His-EB1 and the GFP-Src immunoprecipitate, in the absence (Mock) or presence of λPPase, and analyzed by immunoblotting. (F) In vitro kinase assays were performed with purified His-EB1 and purified GST or GST-Src and analyzed by immunoblotting. (G) HEK293T cells were transfected with GFP-Src or the GFP vector. Immunoprecipitation and immunoblotting were then performed with the indicated antibodies. (H) Purified His-EB1 was incubated with purified GST or GST-Src. GST pulldown and immunoblotting were then performed as indicated. (I) The lysate of HUVECs was immunoprecipitated with the EB1 antibody or IgG control. The interaction between endogenous Src and EB1 was then examined by immunoblotting of the precipitates. All experiments were replicated three times.
    Figure Legend Snippet: Src interacts with and phosphorylates EB1 both in cells and in vitro . (A) HEK293T cells were transfected with HA-EB1 and GFP-Src or the GFP vector. Immunoprecipitation (IP) and immunoblotting (IB) were then performed with the indicated antibodies. (B) HEK293T cells were treated with SU6656 (10 μM), PP2 (10 μM), or equal amount of the vehicle DMSO for 12 h. Immunoprecipitation and immunoblotting were then performed as indicated. (C) In vitro kinase assays were performed with purified His-EB1 and the GFP or GFP-Src immunoprecipitate from HEK293T cells, and examined by immunoblotting. (D) In vitro kinase assays were performed with purified His-EB1 and the GFP-Src immunoprecipitate, in the absence (Mock) or presence of SU6656 (50 μM), PP2 (50 μM), or equal amount of DMSO, and analyzed by immunoblotting. (E) In vitro kinase assays were performed with purified His-EB1 and the GFP-Src immunoprecipitate, in the absence (Mock) or presence of λPPase, and analyzed by immunoblotting. (F) In vitro kinase assays were performed with purified His-EB1 and purified GST or GST-Src and analyzed by immunoblotting. (G) HEK293T cells were transfected with GFP-Src or the GFP vector. Immunoprecipitation and immunoblotting were then performed with the indicated antibodies. (H) Purified His-EB1 was incubated with purified GST or GST-Src. GST pulldown and immunoblotting were then performed as indicated. (I) The lysate of HUVECs was immunoprecipitated with the EB1 antibody or IgG control. The interaction between endogenous Src and EB1 was then examined by immunoblotting of the precipitates. All experiments were replicated three times.

    Techniques Used: In Vitro, Transfection, Plasmid Preparation, Immunoprecipitation, Purification, Incubation

    52) Product Images from "Src-like adaptor protein down-regulates T cell receptor (TCR)-CD3 expression by targeting TCR? for degradation"

    Article Title: Src-like adaptor protein down-regulates T cell receptor (TCR)-CD3 expression by targeting TCR? for degradation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200501164

    SLAP interacts with phospho-TCRζ via the SLAP SH2 domain. (A) Immunoprecipitates of endogenous TCRζ from Jurkat T cells transiently transfected with GFP (V), SLAP-GFP (WT), or SH2-GFP (SH2*) as assessed by Western blot analysis. Half of each transfection was pretreated with the Src family kinase inhibitor PP2 before immunoprecipitation (IP). WCL, whole cell lysate. (B) Immunoprecipitates of TCRζ from JCaM1 (Lck−/−) or JCaM1 stably reconstituted with Lck, as assessed by Western blot analysis. Cell lines were transiently transfected with either GFP or WT-GFP. Dividing lines have been placed to indicate where irrelevant lanes were deleted from the figure. Phospho-TCRζ observed in A and B reflects basal TCRζ phosphorylation that is present in unstimulated cells. (C) Immunoprecipitates of TCRζ from the ZAP-70−/− Jurkat T cell line P116 transfected with GFP or SLAP-GFP, as assessed by Western blot analysis. (D) Localization of GFP fluorescence (green) and endogenous TCRζ (red) in Jurkat T cells that were transiently transfected with GFP, SLAP-GFP, or SH2-GFP as analyzed by deconvolution microscopy. Data are representative of ≥10 cells analyzed per transfection for three independent experiments. (E) MFI of CD3ɛ expression on ZAP-70−/− DP thymocytes incubated in hypertonic medium. Data are the mean of three mice of each genotype ± SEM. Thymocytes from one WT and one SLAP−/− mouse were used for comparison. (F) FACS analysis of CD3ɛ expression on DP and SP thymocytes in the presence (TCRζ Tg) or absence (TCRζ D67–150) of most of the TCRζ cytoplasmic domain. Histograms are representative of three mice per genotype. (G) MFI of CD3ɛ expression on DP and SP thymocytes expressing full-length (TCRζ Tg) or a cytoplasmic truncation (TCRζ D67–150) of the TCRζ cytoplasmic domain that was incubated in hypertonic medium (as assessed by FACS). Data are the mean of three mice per genotype ± SEM.
    Figure Legend Snippet: SLAP interacts with phospho-TCRζ via the SLAP SH2 domain. (A) Immunoprecipitates of endogenous TCRζ from Jurkat T cells transiently transfected with GFP (V), SLAP-GFP (WT), or SH2-GFP (SH2*) as assessed by Western blot analysis. Half of each transfection was pretreated with the Src family kinase inhibitor PP2 before immunoprecipitation (IP). WCL, whole cell lysate. (B) Immunoprecipitates of TCRζ from JCaM1 (Lck−/−) or JCaM1 stably reconstituted with Lck, as assessed by Western blot analysis. Cell lines were transiently transfected with either GFP or WT-GFP. Dividing lines have been placed to indicate where irrelevant lanes were deleted from the figure. Phospho-TCRζ observed in A and B reflects basal TCRζ phosphorylation that is present in unstimulated cells. (C) Immunoprecipitates of TCRζ from the ZAP-70−/− Jurkat T cell line P116 transfected with GFP or SLAP-GFP, as assessed by Western blot analysis. (D) Localization of GFP fluorescence (green) and endogenous TCRζ (red) in Jurkat T cells that were transiently transfected with GFP, SLAP-GFP, or SH2-GFP as analyzed by deconvolution microscopy. Data are representative of ≥10 cells analyzed per transfection for three independent experiments. (E) MFI of CD3ɛ expression on ZAP-70−/− DP thymocytes incubated in hypertonic medium. Data are the mean of three mice of each genotype ± SEM. Thymocytes from one WT and one SLAP−/− mouse were used for comparison. (F) FACS analysis of CD3ɛ expression on DP and SP thymocytes in the presence (TCRζ Tg) or absence (TCRζ D67–150) of most of the TCRζ cytoplasmic domain. Histograms are representative of three mice per genotype. (G) MFI of CD3ɛ expression on DP and SP thymocytes expressing full-length (TCRζ Tg) or a cytoplasmic truncation (TCRζ D67–150) of the TCRζ cytoplasmic domain that was incubated in hypertonic medium (as assessed by FACS). Data are the mean of three mice per genotype ± SEM.

    Techniques Used: Transfection, Western Blot, Immunoprecipitation, Stable Transfection, Fluorescence, Microscopy, Expressing, Incubation, Mouse Assay, FACS

    53) Product Images from "A biosensor generated via high throughput screening quantifies cell edge Src dynamics"

    Article Title: A biosensor generated via high throughput screening quantifies cell edge Src dynamics

    Journal: Nature chemical biology

    doi: 10.1038/nchembio.585

    Automated edge analysis and line scans reveals distinct zone of SFK activity that is correlated with protrusion velocity a) SFK activity as a function of microns from the cell edge. SFK activity is localized towards the edge of the cell and is inhibited by PP2 Treatment. For all data points the standard error is less than 0.1%. At 1μm, the difference between the mean normalized intensity ratio, before and after PP2 treatment, is ~0.08 and is statistically significant (p
    Figure Legend Snippet: Automated edge analysis and line scans reveals distinct zone of SFK activity that is correlated with protrusion velocity a) SFK activity as a function of microns from the cell edge. SFK activity is localized towards the edge of the cell and is inhibited by PP2 Treatment. For all data points the standard error is less than 0.1%. At 1μm, the difference between the mean normalized intensity ratio, before and after PP2 treatment, is ~0.08 and is statistically significant (p

    Techniques Used: Activity Assay

    Src activation dynamics in living cells a) DIC (left panel) and ratio images (right panel) of a PDGF-stimulated NIH 3T3 MEF microinjected with the SFK merobody biosensor. Scale baris 20 μm. Note prominent circular dorsal ruffles b) Ratio image of a PTK1 cell microinjected with the biosensor. Scale bar is 20μm. c) DIC (left panels), m-Cerulean (merobody localization, middle panels), and ratio images (right panels) from representative frames of a movie in which a PTK1 cell microinjected with the biosensor was treated with the Src inhibitor PP2. Scale bar is 10 μm.
    Figure Legend Snippet: Src activation dynamics in living cells a) DIC (left panel) and ratio images (right panel) of a PDGF-stimulated NIH 3T3 MEF microinjected with the SFK merobody biosensor. Scale baris 20 μm. Note prominent circular dorsal ruffles b) Ratio image of a PTK1 cell microinjected with the biosensor. Scale bar is 20μm. c) DIC (left panels), m-Cerulean (merobody localization, middle panels), and ratio images (right panels) from representative frames of a movie in which a PTK1 cell microinjected with the biosensor was treated with the Src inhibitor PP2. Scale bar is 10 μm.

    Techniques Used: Activation Assay

    54) Product Images from "The Shc Family Protein Adaptor, Rai, Negatively Regulates T Cell Antigen Receptor Signaling by Inhibiting ZAP-70 Recruitment and Activation"

    Article Title: The Shc Family Protein Adaptor, Rai, Negatively Regulates T Cell Antigen Receptor Signaling by Inhibiting ZAP-70 Recruitment and Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029899

    Rai forms a complex with CD3 and ZAP-70 and clusters at the immunological synapse. A . Immunoblot analysis with anti-phospho-tyrosine or anti-ZAP-70 antibodies of Rai-specific immunoprecipitates from post-nuclear supernatants of the Rai-expressing Jurkat transfectant activated with anti-CD3 mAb for 1.5 min. Where indicated cells were incubated with 20 µM PP2 prior to activation. A control blot of the stripped filter is shown below. B . Immunoblot analysis with anti-ZAP-70 antibodies of Rai-specific immunoprecipitates from post-nuclear supernatants of splenocytes from wild-type (+/+) or Rai −/− (−/−) mice activated with anti-CD3 mAb for 1.5 min. A control blot is shown below. C . Immunoblot analysis with anti-ZAP-70 antibodies of in vitro binding assays carried out on post-nuclear supernatants of the control Jurkat line using GST fusion proteins containing the PTB, SH2 or CH1 domain of Rai, the latter either non-phosphorylated or phosphorylated in vitro with recombinant Lck. Control blots with anti-GST antibodies are shown below. D . Left , Immunoblot analysis with anti-phospho-tyrosine antibodies of Rai-specific immunoprecipitates from post-nuclear supernatants of the Rai-expressing Jurkat transfectant activated with anti-CD3 mAb for 1.5 min. Right , Immunoblot analysis with anti-Rai antibodies of in vitro binding assays carried out on post-nuclear supernatants of control or Rai-expressing Jurkat cells using a GST fusion protein containing the intracellular domain of human CD3, either non-phosphorylated or phosphorylated in vitro with recombinant Lck. A control blot of the stripped filter is shown below. E . Immunofluorescence analysis of Rai in conjugates of Rai-expressing Jurkat cells and antigen-pulsed APC (SEE), incubated at 37°C for 15 min. Conjugates formed in the absence of antigen are shown on top part of the panel. Representative images are shown. No aspecific fluorescence was observed when Rai-expressing cells were immunostained with secondary antibody alone (not shown).
    Figure Legend Snippet: Rai forms a complex with CD3 and ZAP-70 and clusters at the immunological synapse. A . Immunoblot analysis with anti-phospho-tyrosine or anti-ZAP-70 antibodies of Rai-specific immunoprecipitates from post-nuclear supernatants of the Rai-expressing Jurkat transfectant activated with anti-CD3 mAb for 1.5 min. Where indicated cells were incubated with 20 µM PP2 prior to activation. A control blot of the stripped filter is shown below. B . Immunoblot analysis with anti-ZAP-70 antibodies of Rai-specific immunoprecipitates from post-nuclear supernatants of splenocytes from wild-type (+/+) or Rai −/− (−/−) mice activated with anti-CD3 mAb for 1.5 min. A control blot is shown below. C . Immunoblot analysis with anti-ZAP-70 antibodies of in vitro binding assays carried out on post-nuclear supernatants of the control Jurkat line using GST fusion proteins containing the PTB, SH2 or CH1 domain of Rai, the latter either non-phosphorylated or phosphorylated in vitro with recombinant Lck. Control blots with anti-GST antibodies are shown below. D . Left , Immunoblot analysis with anti-phospho-tyrosine antibodies of Rai-specific immunoprecipitates from post-nuclear supernatants of the Rai-expressing Jurkat transfectant activated with anti-CD3 mAb for 1.5 min. Right , Immunoblot analysis with anti-Rai antibodies of in vitro binding assays carried out on post-nuclear supernatants of control or Rai-expressing Jurkat cells using a GST fusion protein containing the intracellular domain of human CD3, either non-phosphorylated or phosphorylated in vitro with recombinant Lck. A control blot of the stripped filter is shown below. E . Immunofluorescence analysis of Rai in conjugates of Rai-expressing Jurkat cells and antigen-pulsed APC (SEE), incubated at 37°C for 15 min. Conjugates formed in the absence of antigen are shown on top part of the panel. Representative images are shown. No aspecific fluorescence was observed when Rai-expressing cells were immunostained with secondary antibody alone (not shown).

    Techniques Used: Expressing, Transfection, Incubation, Activation Assay, Mouse Assay, In Vitro, Binding Assay, Recombinant, Immunofluorescence, Fluorescence

    55) Product Images from "Fyn Tyrosine Kinase Increases Apolipoprotein E Receptor 2 Levels and Phosphorylation"

    Article Title: Fyn Tyrosine Kinase Increases Apolipoprotein E Receptor 2 Levels and Phosphorylation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0110845

    Fyn’s effects on ApoER2 levels depend on Fyn’s kinase activity. A. COS7 cells were co-transfected with ApoER2 and either Fyn or empty vector and treated with PP2 or DMSO (control) for 6 hours. Lysates were collected in RIPA buffer, Western blotted and probed for ApoER2, Fyn, and tubulin. ** p
    Figure Legend Snippet: Fyn’s effects on ApoER2 levels depend on Fyn’s kinase activity. A. COS7 cells were co-transfected with ApoER2 and either Fyn or empty vector and treated with PP2 or DMSO (control) for 6 hours. Lysates were collected in RIPA buffer, Western blotted and probed for ApoER2, Fyn, and tubulin. ** p

    Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Western Blot

    56) Product Images from "FAK competes for Src to promote migration against invasion in melanoma cells"

    Article Title: FAK competes for Src to promote migration against invasion in melanoma cells

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.329

    Src activity is necessary for invadopodia formation. ( a ) B16F10 cells transiently transfected with control siRNA or FAK siRNA were cultured on Alexa fluor 405 gelatin coverslips and treated or not with the Src inhibitor PP2. No degradation was observed in both control (data not shown) and cells treated with FAK siRNA after Src inhibition. Insets show magnified views of invadopodia. Arrows indicate the colocalization of actin and cortactin at dot-like invadopodia. Scale bar: 20 μ m. ( b ) Cellular extracts were analyzed by western blotting and probed for phosphor-Tyr416-Src. The Graph shows no increase in Src activity after FAK depletion
    Figure Legend Snippet: Src activity is necessary for invadopodia formation. ( a ) B16F10 cells transiently transfected with control siRNA or FAK siRNA were cultured on Alexa fluor 405 gelatin coverslips and treated or not with the Src inhibitor PP2. No degradation was observed in both control (data not shown) and cells treated with FAK siRNA after Src inhibition. Insets show magnified views of invadopodia. Arrows indicate the colocalization of actin and cortactin at dot-like invadopodia. Scale bar: 20 μ m. ( b ) Cellular extracts were analyzed by western blotting and probed for phosphor-Tyr416-Src. The Graph shows no increase in Src activity after FAK depletion

    Techniques Used: Activity Assay, Transfection, Cell Culture, Inhibition, Western Blot

    57) Product Images from "Activation of Cdc42 by trans interactions of the cell adhesion molecules nectins through c-Src and Cdc42-GEF FRG"

    Article Title: Activation of Cdc42 by trans interactions of the cell adhesion molecules nectins through c-Src and Cdc42-GEF FRG

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200401093

    Nectin-induced, c-Src– mediated activation of FRG. (A) Recruitment of FRG to the contact sites between the Nef-3–coated beads and nectin-1-L cells. Nectin-1-L or EL cells transiently overexpressing GFP-FRG were incubated with the Nef-3–, ConA-, or Cef-coated beads for 1 h and stained with the anti–nectin-1 pAb. The signal for E-cadherin–β-catenin complex was detected by the anti–β-catenin mAb. (a) Nectin-1-L cells; (b) EL cells. Bars, 10 μm. Positions of the beads are marked with asterisks. The inset indicates the magnified image of the boxed area. Bars in the quantitative analysis of panels a and b represent the percentage of the bead–cell contact sites with the signal for nectin-1 or E-cadherin of the total bead–cell contact sites counted ( n = 50) or the percentage of the bead–cell contact sites with the signal for GFP of the bead–cell contact sites with the signal for nectin-1 or E-cadherin counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments. (B) Nectin-induced tyrosine phosphorylation of FRG. (a) Wild-type L, nectin-1-L, or EL cells transiently overexpressing Flag-FRG and c-Src were treated with clustered Nef-3 or IgG in the presence or absence of PP2 for indicated periods of time. (b) Nectin-1-L cells transiently overexpressing c-Src and Flag-FRG or Flag-frabin were treated with clustered Nef-3 or IgG for 30 min. Each cell lysate was subjected to the immunoprecipitation assay with the anti-Flag mAb, followed by Western blotting with the anti-phosphotyrosine, anti-Flag, and anti–v-Src mAbs. (C) GEF activity of FRG or frabin on Cdc42. The binding of [ 35 S]GTPγS to Cdc42 was assayed by incubation with the immunoprecipitant prepared in B for indicated periods of time. The results shown are representative of three independent experiments.
    Figure Legend Snippet: Nectin-induced, c-Src– mediated activation of FRG. (A) Recruitment of FRG to the contact sites between the Nef-3–coated beads and nectin-1-L cells. Nectin-1-L or EL cells transiently overexpressing GFP-FRG were incubated with the Nef-3–, ConA-, or Cef-coated beads for 1 h and stained with the anti–nectin-1 pAb. The signal for E-cadherin–β-catenin complex was detected by the anti–β-catenin mAb. (a) Nectin-1-L cells; (b) EL cells. Bars, 10 μm. Positions of the beads are marked with asterisks. The inset indicates the magnified image of the boxed area. Bars in the quantitative analysis of panels a and b represent the percentage of the bead–cell contact sites with the signal for nectin-1 or E-cadherin of the total bead–cell contact sites counted ( n = 50) or the percentage of the bead–cell contact sites with the signal for GFP of the bead–cell contact sites with the signal for nectin-1 or E-cadherin counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments. (B) Nectin-induced tyrosine phosphorylation of FRG. (a) Wild-type L, nectin-1-L, or EL cells transiently overexpressing Flag-FRG and c-Src were treated with clustered Nef-3 or IgG in the presence or absence of PP2 for indicated periods of time. (b) Nectin-1-L cells transiently overexpressing c-Src and Flag-FRG or Flag-frabin were treated with clustered Nef-3 or IgG for 30 min. Each cell lysate was subjected to the immunoprecipitation assay with the anti-Flag mAb, followed by Western blotting with the anti-phosphotyrosine, anti-Flag, and anti–v-Src mAbs. (C) GEF activity of FRG or frabin on Cdc42. The binding of [ 35 S]GTPγS to Cdc42 was assayed by incubation with the immunoprecipitant prepared in B for indicated periods of time. The results shown are representative of three independent experiments.

    Techniques Used: Activation Assay, Incubation, Staining, Immunoprecipitation, Western Blot, Activity Assay, Binding Assay

    Involvement of SFKs in the nectin-induced formation of the E-cadherin–based AJs in MDCK cells. (A and B) Inhibitory effects of PP2 on the nectin-induced, E-cadherin–based formation of AJs in MDCK cells. Wild-type MDCK, nectin-1-MDCK, and nectin-1-MDCK cells overexpressing GFP-V12Cdc42 or GFP were incubated at 2 μM Ca 2+ for 2 h and then incubated at 2 mM Ca 2+ in the presence of PP2 or PP3 for 2 h. After the incubation, the cells were immunostained with the anti–E-cadherin mAb. Bars, 10 μm. (A) Wild-type MDCK and nectin-1-MDCK cells in the presence of PP2 or PP3. Arrows show the absence of the signal for E-cadherin at the cell–cell adhesion sites. (B) Nectin-1-MDCK cells expressing GFP-V12Cdc42 or GFP in the presence of PP2. Arrowheads show the presence or absence of the signal for E-cadherin at the cell–cell adhesion site. Bars in the quantitative analysis of A and B represent the percentage of cell–cell adhesions with the signal for E-cadherin of the total cell–cell adhesions counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments.
    Figure Legend Snippet: Involvement of SFKs in the nectin-induced formation of the E-cadherin–based AJs in MDCK cells. (A and B) Inhibitory effects of PP2 on the nectin-induced, E-cadherin–based formation of AJs in MDCK cells. Wild-type MDCK, nectin-1-MDCK, and nectin-1-MDCK cells overexpressing GFP-V12Cdc42 or GFP were incubated at 2 μM Ca 2+ for 2 h and then incubated at 2 mM Ca 2+ in the presence of PP2 or PP3 for 2 h. After the incubation, the cells were immunostained with the anti–E-cadherin mAb. Bars, 10 μm. (A) Wild-type MDCK and nectin-1-MDCK cells in the presence of PP2 or PP3. Arrows show the absence of the signal for E-cadherin at the cell–cell adhesion sites. (B) Nectin-1-MDCK cells expressing GFP-V12Cdc42 or GFP in the presence of PP2. Arrowheads show the presence or absence of the signal for E-cadherin at the cell–cell adhesion site. Bars in the quantitative analysis of A and B represent the percentage of cell–cell adhesions with the signal for E-cadherin of the total cell–cell adhesions counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments.

    Techniques Used: Incubation, Expressing

    Inhibition by PP2 and Csk of the nectin-induced formation of filopodia and lamellipodia in nectin-1-L cells. (A) Nectin-induced formation of filopodia and lamellipodia. Nectin-1-L or wild-type L cells were cultured on the Nef-3–, IgG-, or PLL-coated coverslips for 30 min and stained for F-actin with rhodamine-phalloidin. Bars in the quantitative analysis represent the number of cells attached on the coverslips per millimeter squared and the percentage of cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments. (B) Inhibitory effect of PP2. Nectin-1-L cells were cultured on the Nef-3– or IgG-coated coverslips in the presence of PP2, PP3, or DMSO for 30 min and stained with rhodamine-phalloidin. (C) Inhibitory effect of Csk. Nectin-1-L cells infected with Av1CATcsk or Av1CATlacZ were cultured on the Nef-3–coated coverslips for 30 min and stained with rhodamine-phalloidin and the anti-Csk or the anti–β-galactosidase mAbs, respectively. β-Gal, β-galactosidase. Bars in the quantitative analysis of B and C represent the percentage of cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of three independent experiments. Bars, 10 μm.
    Figure Legend Snippet: Inhibition by PP2 and Csk of the nectin-induced formation of filopodia and lamellipodia in nectin-1-L cells. (A) Nectin-induced formation of filopodia and lamellipodia. Nectin-1-L or wild-type L cells were cultured on the Nef-3–, IgG-, or PLL-coated coverslips for 30 min and stained for F-actin with rhodamine-phalloidin. Bars in the quantitative analysis represent the number of cells attached on the coverslips per millimeter squared and the percentage of cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments. (B) Inhibitory effect of PP2. Nectin-1-L cells were cultured on the Nef-3– or IgG-coated coverslips in the presence of PP2, PP3, or DMSO for 30 min and stained with rhodamine-phalloidin. (C) Inhibitory effect of Csk. Nectin-1-L cells infected with Av1CATcsk or Av1CATlacZ were cultured on the Nef-3–coated coverslips for 30 min and stained with rhodamine-phalloidin and the anti-Csk or the anti–β-galactosidase mAbs, respectively. β-Gal, β-galactosidase. Bars in the quantitative analysis of B and C represent the percentage of cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of three independent experiments. Bars, 10 μm.

    Techniques Used: Inhibition, Cell Culture, Staining, Infection

    Inhibition by PP2 of the nectin-induced activation of Cdc42 in nectin-1-L cells. (A and B) FRET imaging. Nectin-1-L cells expressing Raichu-Cdc42 were cultured on the Nef-3–coated dishes in the presence of PP2 (A) or PP3 (B) for 1 h, and FRET imaging was obtained at indicated periods of time. The upper and lower limits of the YFP/CFP ratio range are shown. Arrowheads indicate the position of filopodia. Bars, 10 μm. The results shown are representative of ten independent experiments. (C) Pull-down assay. Nectin-1-L cells expressing c-Src and GFP-Cdc42 were cultured with clustered Nef-3 or IgG in the presence or absence of PP2 or PP3 for indicated periods of time and then subjected to the pull-down assay. The results shown are representative of three independent experiments.
    Figure Legend Snippet: Inhibition by PP2 of the nectin-induced activation of Cdc42 in nectin-1-L cells. (A and B) FRET imaging. Nectin-1-L cells expressing Raichu-Cdc42 were cultured on the Nef-3–coated dishes in the presence of PP2 (A) or PP3 (B) for 1 h, and FRET imaging was obtained at indicated periods of time. The upper and lower limits of the YFP/CFP ratio range are shown. Arrowheads indicate the position of filopodia. Bars, 10 μm. The results shown are representative of ten independent experiments. (C) Pull-down assay. Nectin-1-L cells expressing c-Src and GFP-Cdc42 were cultured with clustered Nef-3 or IgG in the presence or absence of PP2 or PP3 for indicated periods of time and then subjected to the pull-down assay. The results shown are representative of three independent experiments.

    Techniques Used: Inhibition, Activation Assay, Imaging, Expressing, Cell Culture, Pull Down Assay

    Suppression by V12Cdc42 and V12Rac1 of the inhibitory effect of PP2 in nectin-1-L cells. Nectin-1-L cells transiently overexpressing GFP-V12Cdc42, GFP-V12Rac1, or GFP were cultured on the Nef-3–coated coverslips in the presence of PP2 or PP3 for 1 h, and stained for F-actin with rhodamine-phalloidin. Bars, 10 μm. Bars in the quantitative analysis represent the percentage of cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments.
    Figure Legend Snippet: Suppression by V12Cdc42 and V12Rac1 of the inhibitory effect of PP2 in nectin-1-L cells. Nectin-1-L cells transiently overexpressing GFP-V12Cdc42, GFP-V12Rac1, or GFP were cultured on the Nef-3–coated coverslips in the presence of PP2 or PP3 for 1 h, and stained for F-actin with rhodamine-phalloidin. Bars, 10 μm. Bars in the quantitative analysis represent the percentage of cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments.

    Techniques Used: Cell Culture, Staining

    Inhibition by PP2 and Csk of the nectin-induced formation of filopodia and lamellipodia in nectin-1-MDCK cells. (A) Nectin-induced formation of filopodia and lamellipodia. Nectin-1-MDCK or wild-type MDCK cells were cultured on the Nef-3–, IgG-, Cef-, or PLL-coated coverslips for 2 h and stained for F-actin with rhodamine-phalloidin. Bars in the quantitative analysis represent number of the cells attached on the coverslips per millimeter squared and percentage of the cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments. (B) Inhibitory effect of PP2. Nectin-1-MDCK cells were cultured on the Nef-3– or IgG-coated coverslips in the presence of PP2, PP3, or DMSO for 2 h and stained with rhodamine-phalloidin. (C) Inhibitory effect of Csk. Nectin-1-MDCK cells infected with Av1CATcsk or Av1CATlacZ were cultured on the Nef-3–coated coverslips for 2 h and stained with rhodamine-phalloidin and the anti-Csk or the anti–β-galactosidase mAb, respectively. β-Gal, β-galactosidase. Bars in the quantitative analysis of B and C represent the percentage of cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments. Bars, 10 μm.
    Figure Legend Snippet: Inhibition by PP2 and Csk of the nectin-induced formation of filopodia and lamellipodia in nectin-1-MDCK cells. (A) Nectin-induced formation of filopodia and lamellipodia. Nectin-1-MDCK or wild-type MDCK cells were cultured on the Nef-3–, IgG-, Cef-, or PLL-coated coverslips for 2 h and stained for F-actin with rhodamine-phalloidin. Bars in the quantitative analysis represent number of the cells attached on the coverslips per millimeter squared and percentage of the cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments. (B) Inhibitory effect of PP2. Nectin-1-MDCK cells were cultured on the Nef-3– or IgG-coated coverslips in the presence of PP2, PP3, or DMSO for 2 h and stained with rhodamine-phalloidin. (C) Inhibitory effect of Csk. Nectin-1-MDCK cells infected with Av1CATcsk or Av1CATlacZ were cultured on the Nef-3–coated coverslips for 2 h and stained with rhodamine-phalloidin and the anti-Csk or the anti–β-galactosidase mAb, respectively. β-Gal, β-galactosidase. Bars in the quantitative analysis of B and C represent the percentage of cells with filopodia and/or lamellipodia of the total cells counted ( n = 50) and are expressed as means ± SEMs of the three independent experiments. Bars, 10 μm.

    Techniques Used: Inhibition, Cell Culture, Staining, Infection

    58) Product Images from "The CaV2α1 EF-hand F helix tyrosine, a highly conserved locus for GPCR inhibition of CaV2 channels"

    Article Title: The CaV2α1 EF-hand F helix tyrosine, a highly conserved locus for GPCR inhibition of CaV2 channels

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-21586-5

    Inhibition of the sensory neuron single action potential Fluo-4 transient with 5HT1A expression activation is inhibited with Src kinase inhibition or point mutation of the Ca V 2α1 EF-hand F-helix tyrosine (Y1501F). ( A ) Images are RFP- Ca V 2α1, Fluo-4 load with ROI selected, and Fluo-4 frame 74 before an action potential from frame 75 after an action potential approximately 50 ms later. On the far right is raw data for three single action potentials measured at the ROI marked in the middle image in white. The 20 μm scale bar on left panel applies to all the images. ( B ) Average of all single action potential Fluo-4 transients before and after application of 8-OH-DPAT for the five groups measured at 48 h and the one group 24 h post expression vector injection compared in CDE. The after measurement was made 30 s-1m after application of the 5HT1A agonist 8-OH-DPAT, whenever the change in membrane excitability was noted, and at one minute for the no injection group that do not respond to the agonist. Comparison of the initial peak Fluo-4 transient amplitudes (ΔF/F No Inj 0.089 ± 0.14, 1A 0.081 ± 0.018, wt + 1A 0.099 ± 0.023, Y/F + 1A 24 h 0.098 ± 0.031, Y/F + 1A 48 h 0.053 ± 0.008, wt + 1A + PP2 0.106 ± 0.009) between the different groups and the No Injection controls did not find any significant differences (a one-way ANOVA F = 0.731 and Dunnett’s post-test was used to compare groups to the No Injection control, P = 0.763, 0.921, 0.907, 0.373, 0.952, n = 7,6,7,5,7,7). ( C ) Summary of the change in the average single action potential peak Fluo-4 transient after 8-OH-DPAT application for the six different conditions. Five groups were measured after 48 h expression; No Injection, RFP + 5HT1A, RFP-Ca V 2α1 wt + 5HT1A, RFP-Ca V 2α1 Y1501F + 5HT1A, and a −20 min preincubation with PP2 and RFP-Ca V 2α1 wt + 5HT1A. Another group was measured after only 24 h expression of RFP-Ca V 2α1 Y1501F + 5HT1A. Groups expressing the 5HT1A receptor all had significantly (***P
    Figure Legend Snippet: Inhibition of the sensory neuron single action potential Fluo-4 transient with 5HT1A expression activation is inhibited with Src kinase inhibition or point mutation of the Ca V 2α1 EF-hand F-helix tyrosine (Y1501F). ( A ) Images are RFP- Ca V 2α1, Fluo-4 load with ROI selected, and Fluo-4 frame 74 before an action potential from frame 75 after an action potential approximately 50 ms later. On the far right is raw data for three single action potentials measured at the ROI marked in the middle image in white. The 20 μm scale bar on left panel applies to all the images. ( B ) Average of all single action potential Fluo-4 transients before and after application of 8-OH-DPAT for the five groups measured at 48 h and the one group 24 h post expression vector injection compared in CDE. The after measurement was made 30 s-1m after application of the 5HT1A agonist 8-OH-DPAT, whenever the change in membrane excitability was noted, and at one minute for the no injection group that do not respond to the agonist. Comparison of the initial peak Fluo-4 transient amplitudes (ΔF/F No Inj 0.089 ± 0.14, 1A 0.081 ± 0.018, wt + 1A 0.099 ± 0.023, Y/F + 1A 24 h 0.098 ± 0.031, Y/F + 1A 48 h 0.053 ± 0.008, wt + 1A + PP2 0.106 ± 0.009) between the different groups and the No Injection controls did not find any significant differences (a one-way ANOVA F = 0.731 and Dunnett’s post-test was used to compare groups to the No Injection control, P = 0.763, 0.921, 0.907, 0.373, 0.952, n = 7,6,7,5,7,7). ( C ) Summary of the change in the average single action potential peak Fluo-4 transient after 8-OH-DPAT application for the six different conditions. Five groups were measured after 48 h expression; No Injection, RFP + 5HT1A, RFP-Ca V 2α1 wt + 5HT1A, RFP-Ca V 2α1 Y1501F + 5HT1A, and a −20 min preincubation with PP2 and RFP-Ca V 2α1 wt + 5HT1A. Another group was measured after only 24 h expression of RFP-Ca V 2α1 Y1501F + 5HT1A. Groups expressing the 5HT1A receptor all had significantly (***P

    Techniques Used: Inhibition, Expressing, Activation Assay, Mutagenesis, Mass Spectrometry, Plasmid Preparation, Injection

    59) Product Images from "Non-Catalytic Functions of Pyk2 and Fyn Regulate Late Stage Adhesion in Human T Cells"

    Article Title: Non-Catalytic Functions of Pyk2 and Fyn Regulate Late Stage Adhesion in Human T Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0053011

    The enzymatic activity of the Src family kinases is needed for early but not late TCR-mediated adhesion. (A) Jurkat E6.1 cells (left panel) or hAPBTs (right panel) were stained and then incubated with 25 µM of the Src family kinase inhibitor PP2 for 15 minutes at 37°C. The cells were then stimulated on an anti-TCR coated RIA/EIA plate for various times. The data were normalized such that the 30 minute time point was set to 100%. The mean ± SD from three to four independent replicates for each stimulation time was then calculated and graphed. * p≤0.05 ** p≤0.01 (B) Jurkat E6.1 cells and hAPBTs were stained and then incubated with DMSO or the indicated doses of PP2 for 15 minutes. The cells were then stimulated with immobilized anti-TCR for 30 minutes. The amount of cellular binding was normalized to the DMSO sample. The Jurkat E6.1 graph shows the average of six independent experiments ± SD, and the hAPBT results are the mean of three independent experiments ± SD. * p≤0.05 (C) Jurkat E6.1 cells (left) and hAPBTs (right) were incubated with various doses of PP2 for 15 minutes and then stimulated with soluble anti-TCR antibody for five minutes or left unstimulated (US) as a control. Cellular lysates were resolved by PAGE and total changes in TCR-inducible tyrosine phosphorylation or Pyk2 tyrosine 402 phosphorylation were detected by immunoblotting. Actin was used as a loading control. (D) Jurkat cells or hAPBTs were stained and treated with PP2 for 15 minutes after beginning TCR stimulation using immobilized antibodies. After 30 minutes, the amount of adhesion was determined and normalized to the DMSO samples. The graph shows the average of three independent experiments ± SD.
    Figure Legend Snippet: The enzymatic activity of the Src family kinases is needed for early but not late TCR-mediated adhesion. (A) Jurkat E6.1 cells (left panel) or hAPBTs (right panel) were stained and then incubated with 25 µM of the Src family kinase inhibitor PP2 for 15 minutes at 37°C. The cells were then stimulated on an anti-TCR coated RIA/EIA plate for various times. The data were normalized such that the 30 minute time point was set to 100%. The mean ± SD from three to four independent replicates for each stimulation time was then calculated and graphed. * p≤0.05 ** p≤0.01 (B) Jurkat E6.1 cells and hAPBTs were stained and then incubated with DMSO or the indicated doses of PP2 for 15 minutes. The cells were then stimulated with immobilized anti-TCR for 30 minutes. The amount of cellular binding was normalized to the DMSO sample. The Jurkat E6.1 graph shows the average of six independent experiments ± SD, and the hAPBT results are the mean of three independent experiments ± SD. * p≤0.05 (C) Jurkat E6.1 cells (left) and hAPBTs (right) were incubated with various doses of PP2 for 15 minutes and then stimulated with soluble anti-TCR antibody for five minutes or left unstimulated (US) as a control. Cellular lysates were resolved by PAGE and total changes in TCR-inducible tyrosine phosphorylation or Pyk2 tyrosine 402 phosphorylation were detected by immunoblotting. Actin was used as a loading control. (D) Jurkat cells or hAPBTs were stained and treated with PP2 for 15 minutes after beginning TCR stimulation using immobilized antibodies. After 30 minutes, the amount of adhesion was determined and normalized to the DMSO samples. The graph shows the average of three independent experiments ± SD.

    Techniques Used: Activity Assay, Staining, Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Polyacrylamide Gel Electrophoresis

    60) Product Images from "Effects of Progesterone and Medroxyprogesterone on Actin Remodeling and Neuronal Spine Formation"

    Article Title: Effects of Progesterone and Medroxyprogesterone on Actin Remodeling and Neuronal Spine Formation

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2012-1278

    PR Signaling Cascades to Moesin, FAK, and WAVE1 A, Neurons were exposed to 1 nM P or MPA for 20 minutes, in the presence or absence of the Src kinase inhibitor, PP2 (10 μM), after transfection with a dominant-negative Rac1 (RAC1TN17DN) construct, or of the specific Cdk5 inhibitor, roscovitine (Rosc; 50 μM) or B) silencing of ROCK2 with siRNAs (panel B) or with a specific inhibitor of FAK (panel D) (FAKi, 1 μM). A, B, and D, Neuronal cell content of c-Src, Rac1, Cdk5, ROCK2, moesin, FAK, and WAVE1 or of the relative phosphorylated forms were assayed with Western analysis. C and E, Cells were treated with P or MPA (both 1 nM) for 20 minutes in the presence or absence of the ROCK inhibitor, Y-27632 (Y - 10 μM) (panel C) or with a specific inhibitor of FAK (FAKi, 1 μM) (panel D). C and E, ROCK-2 was immunoprecipitated and the IPs were used to phosphorylate the bait protein, MBP. ROCK-2 kinase activity is shown as the amount of phosphorylated MBP (P-MBP). All experiments were performed in triplicates and representative images are shown. Densitometric quantifications of all the blots (including those not shown) were performed, and the relative mean ± SD of each condition is presented in a graph as supplemental data. CON, control.
    Figure Legend Snippet: PR Signaling Cascades to Moesin, FAK, and WAVE1 A, Neurons were exposed to 1 nM P or MPA for 20 minutes, in the presence or absence of the Src kinase inhibitor, PP2 (10 μM), after transfection with a dominant-negative Rac1 (RAC1TN17DN) construct, or of the specific Cdk5 inhibitor, roscovitine (Rosc; 50 μM) or B) silencing of ROCK2 with siRNAs (panel B) or with a specific inhibitor of FAK (panel D) (FAKi, 1 μM). A, B, and D, Neuronal cell content of c-Src, Rac1, Cdk5, ROCK2, moesin, FAK, and WAVE1 or of the relative phosphorylated forms were assayed with Western analysis. C and E, Cells were treated with P or MPA (both 1 nM) for 20 minutes in the presence or absence of the ROCK inhibitor, Y-27632 (Y - 10 μM) (panel C) or with a specific inhibitor of FAK (FAKi, 1 μM) (panel D). C and E, ROCK-2 was immunoprecipitated and the IPs were used to phosphorylate the bait protein, MBP. ROCK-2 kinase activity is shown as the amount of phosphorylated MBP (P-MBP). All experiments were performed in triplicates and representative images are shown. Densitometric quantifications of all the blots (including those not shown) were performed, and the relative mean ± SD of each condition is presented in a graph as supplemental data. CON, control.

    Techniques Used: Transfection, Dominant Negative Mutation, Construct, Western Blot, Immunoprecipitation, Activity Assay

    61) Product Images from "CD28 plays a critical role in the segregation of PKC? within the immunologic synapse"

    Article Title: CD28 plays a critical role in the segregation of PKC? within the immunologic synapse

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.142298399

    TCR-mediated PCKθ capping is Lck-dependent. ( A ) Naive AD10 CD4 + T cells were pretreated at 37°C for 1 h with vehicle alone (0.1% DMSO) ( a ) or 20 μM PP2 ( b ). The T cells were then incubated with anti-CD3-coated beads for 45 min, after which time T cells were stained for PCKθ. The asterisk indicates the position of the CD3-coated beads. ( B ) Naïve CD4 + T cells were purified from Fyn −/− and LGF + Lck −/− mice, stimulated with anti-CD3-coated beads, fixed, and stained for PKCθ localization. Results are representative of three similar experiments.
    Figure Legend Snippet: TCR-mediated PCKθ capping is Lck-dependent. ( A ) Naive AD10 CD4 + T cells were pretreated at 37°C for 1 h with vehicle alone (0.1% DMSO) ( a ) or 20 μM PP2 ( b ). The T cells were then incubated with anti-CD3-coated beads for 45 min, after which time T cells were stained for PCKθ. The asterisk indicates the position of the CD3-coated beads. ( B ) Naïve CD4 + T cells were purified from Fyn −/− and LGF + Lck −/− mice, stimulated with anti-CD3-coated beads, fixed, and stained for PKCθ localization. Results are representative of three similar experiments.

    Techniques Used: Incubation, Staining, Purification, Mouse Assay

    62) Product Images from "VEGF165-induced vascular permeability requires NRP1 for ABL-mediated SRC family kinase activation"

    Article Title: VEGF165-induced vascular permeability requires NRP1 for ABL-mediated SRC family kinase activation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20160311

    VEGFR2 and NRP1 are required for VEGF165-induced SFK activation via ABL1 . (A–C) Confluent HDMEC cultures transfected with si-control or siNRP1 were serum-starved and treated with VEGF165 for the indicated times. Cultures were also treated with vehicle (-) or PTK/ZK (+) for 30 min before VEGF165 stimulation. Lysates were used for immunoblotting with the indicated antibodies (A), followed by quantification of pSFK levels (B, left) and pCRKL levels (B’, right, and C) relative to GAPDH (four independent experiments). Each of the two vertical lines indicated a group of immunoblots from a single gel, with both gels containing aliquots of the same protein lysate. (D–E) Confluent HDMEC cultures transfected with si-control or siABL1 were serum-starved and treated with VEGF165 for the indicated times. Lysates were used for immunoblotting with the indicated antibodies (D), followed by quantification of pSFK levels (E, left) and pCRKL levels (E’, right) relative to GAPDH (four independent experiments). Each of the two vertical lines indicates a group of immunoblots from a single gel, with both gels containing aliquots of the same protein lysate. The spacer line (D, bottom) separates lanes 4–6 (left) from lanes 1–3 (right) of immunoblots from the gel in Fig. S1. (F–G) Confluent HDMEC cultures were serum-starved and treated with vehicle, Imatinib or PP2 for 30 min before VEGF165 stimulation for the indicated times. Lysates were used for immunoblotting with the indicated antibodies (F), followed by quantification of pSFK levels (G, left) and pCRKL levels (G’, right) relative to GAPDH (three independent experiments). Each of the two vertical lines indicates a group of immunoblots obtained from a single gel, with both gels containing aliquots of the same protein lysate. In B, E, and G (left) data are expressed as fold change, mean ± SEM, in VEGF165-treated cells at 5 and 15 min relative to 0 min; in C and B, E, and G (right), data are expressed as fold change, mean ± SEM, in VEGF165-treated cells at 5 and 15 min relative to control cells at 0 min; asterisks indicate P-values for induction after VEGF165 treatment (*, P
    Figure Legend Snippet: VEGFR2 and NRP1 are required for VEGF165-induced SFK activation via ABL1 . (A–C) Confluent HDMEC cultures transfected with si-control or siNRP1 were serum-starved and treated with VEGF165 for the indicated times. Cultures were also treated with vehicle (-) or PTK/ZK (+) for 30 min before VEGF165 stimulation. Lysates were used for immunoblotting with the indicated antibodies (A), followed by quantification of pSFK levels (B, left) and pCRKL levels (B’, right, and C) relative to GAPDH (four independent experiments). Each of the two vertical lines indicated a group of immunoblots from a single gel, with both gels containing aliquots of the same protein lysate. (D–E) Confluent HDMEC cultures transfected with si-control or siABL1 were serum-starved and treated with VEGF165 for the indicated times. Lysates were used for immunoblotting with the indicated antibodies (D), followed by quantification of pSFK levels (E, left) and pCRKL levels (E’, right) relative to GAPDH (four independent experiments). Each of the two vertical lines indicates a group of immunoblots from a single gel, with both gels containing aliquots of the same protein lysate. The spacer line (D, bottom) separates lanes 4–6 (left) from lanes 1–3 (right) of immunoblots from the gel in Fig. S1. (F–G) Confluent HDMEC cultures were serum-starved and treated with vehicle, Imatinib or PP2 for 30 min before VEGF165 stimulation for the indicated times. Lysates were used for immunoblotting with the indicated antibodies (F), followed by quantification of pSFK levels (G, left) and pCRKL levels (G’, right) relative to GAPDH (three independent experiments). Each of the two vertical lines indicates a group of immunoblots obtained from a single gel, with both gels containing aliquots of the same protein lysate. In B, E, and G (left) data are expressed as fold change, mean ± SEM, in VEGF165-treated cells at 5 and 15 min relative to 0 min; in C and B, E, and G (right), data are expressed as fold change, mean ± SEM, in VEGF165-treated cells at 5 and 15 min relative to control cells at 0 min; asterisks indicate P-values for induction after VEGF165 treatment (*, P

    Techniques Used: Activation Assay, Transfection, Western Blot

    63) Product Images from "?1 Integrin-Focal Adhesion Kinase (FAK) Signaling Modulates Retinal Ganglion Cell (RGC) Survival"

    Article Title: ?1 Integrin-Focal Adhesion Kinase (FAK) Signaling Modulates Retinal Ganglion Cell (RGC) Survival

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0048332

    Schematic diagram of laminin-integrin survival signaling in RGC. RGC adhesion to laminin induces integrin activation and initiates integrin signaling. Integrin β1 undergoes a conformational change to an “active” high affinity bound state [83] . Alternatively, in the absence of laminin, or in the context of MMP-9 mediated laminin degradation observed after RIRI in vivo , a β1 integrin stimulatory antibody can bind and maintain integrin β1 in the “active”, signaling conformation at the RGC’s membrane. Activated β1 integrin initiates signal transduction by recruiting the tyrosine protein kinase, FAK. In response to integrin engagement, FAK auto-phosphorylates at Tyr 397 which represents the initial, major step in FAK activation. FAK P-Tyr 397 creates a binding site for Src family kinases. Src recruitment, and activation leads to phosphorylation of multiple amino acids including Tyr 576, 577 required for FAK’s full catalytic activity [37] . PP2 inhibits Src, therefore FAK’s complete activation required for downstream survival signaling. Initial Tyr 397 phosphorylation is also important for recruitment of PI3K. FAK promotes cell survival downstream of laminin and integrin β1 by enhancing PI3K mediated activation of Akt by phosphorylation at Ser 473. Activated Akt is associated with survival [35] , [37] , and inhibition of apoptosis by increasing expression of anti-apoptotic protein bclx L .
    Figure Legend Snippet: Schematic diagram of laminin-integrin survival signaling in RGC. RGC adhesion to laminin induces integrin activation and initiates integrin signaling. Integrin β1 undergoes a conformational change to an “active” high affinity bound state [83] . Alternatively, in the absence of laminin, or in the context of MMP-9 mediated laminin degradation observed after RIRI in vivo , a β1 integrin stimulatory antibody can bind and maintain integrin β1 in the “active”, signaling conformation at the RGC’s membrane. Activated β1 integrin initiates signal transduction by recruiting the tyrosine protein kinase, FAK. In response to integrin engagement, FAK auto-phosphorylates at Tyr 397 which represents the initial, major step in FAK activation. FAK P-Tyr 397 creates a binding site for Src family kinases. Src recruitment, and activation leads to phosphorylation of multiple amino acids including Tyr 576, 577 required for FAK’s full catalytic activity [37] . PP2 inhibits Src, therefore FAK’s complete activation required for downstream survival signaling. Initial Tyr 397 phosphorylation is also important for recruitment of PI3K. FAK promotes cell survival downstream of laminin and integrin β1 by enhancing PI3K mediated activation of Akt by phosphorylation at Ser 473. Activated Akt is associated with survival [35] , [37] , and inhibition of apoptosis by increasing expression of anti-apoptotic protein bclx L .

    Techniques Used: Activation Assay, In Vivo, Transduction, Binding Assay, Activity Assay, Inhibition, Expressing

    The agonist antibody HUTS-21 mimics laminin’s pro-survival effect in RGC. P5 purified RGCs were cultured on either laminin, or PDL for 48 hours. HUTS-21, or isotype control antibodies (1 µg/ml) were added to RGCs cultured on PDL 12 hours after plating. RGCs were pretreated with the Src/FAK inhibitor PP2 (25 µM). Live-cell imaging with an Axiovert Zeiss 200 M microscope was used to acquire micrographs of RGC in culture immediately after HUTS-21 addition, t = 0, and at the end of the experiment, 48 hours after plating, t = 48 hours. A. To evaluate RGC survival 100–500 live RGC/experimental condition were counted manually in the acquired micrographs to determine the percentage of live RGC at t = 48 hours when compared with t = 0. (N = 6 experiments were performed). B. Average total neurite length per RGC was evaluated by manually measuring RGC neurites in photo-micrographs acquired by live-cell imaging. (N = 6 experiments). Error bars, SD. Student’s t test. **p
    Figure Legend Snippet: The agonist antibody HUTS-21 mimics laminin’s pro-survival effect in RGC. P5 purified RGCs were cultured on either laminin, or PDL for 48 hours. HUTS-21, or isotype control antibodies (1 µg/ml) were added to RGCs cultured on PDL 12 hours after plating. RGCs were pretreated with the Src/FAK inhibitor PP2 (25 µM). Live-cell imaging with an Axiovert Zeiss 200 M microscope was used to acquire micrographs of RGC in culture immediately after HUTS-21 addition, t = 0, and at the end of the experiment, 48 hours after plating, t = 48 hours. A. To evaluate RGC survival 100–500 live RGC/experimental condition were counted manually in the acquired micrographs to determine the percentage of live RGC at t = 48 hours when compared with t = 0. (N = 6 experiments were performed). B. Average total neurite length per RGC was evaluated by manually measuring RGC neurites in photo-micrographs acquired by live-cell imaging. (N = 6 experiments). Error bars, SD. Student’s t test. **p

    Techniques Used: Purification, Cell Culture, Live Cell Imaging, Microscopy

    64) Product Images from "Direct contact with perivascular tumor cells enhances integrin αvβ3 signaling and migration of endothelial cells"

    Article Title: Direct contact with perivascular tumor cells enhances integrin αvβ3 signaling and migration of endothelial cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9700

    The direct interaction of ECs and CSCs promotes EC migration towards bFGF A. Diagrammatic representation of experimental design. Filters (3-μm pore) were coated on both sides with 20 μg/mL laminin. Red-fluorescent-ECs (3×10 4 ) were seeded alone or mixed with GFP-CSCs-(08387) (3×10 4 ) and co-seeded in growth factor-free NBM with 1% BSA on top of the filter. Growth factor-free NBM with 10 ng/ml bFGF was placed in the bottom chamber, and the cells allowed to migrate (37°C, 5% CO 2 ). At 6 h, cells were removed from the upper filter surface and cells on the lower filter surface were washed, fixed, photographed and quantified. B. The Src inhibitor PP2 (1 μM) was incubated with ECs (20 min), followed by washing, and EC seeding alone or co-seeding with CSCs. C. ECs were seeded in CM/EC+CSC (described in Figure 2B ), or co-seeded with CSCs. D, E. ECs were treated with integrin β3 specific siRNA, L1CAM specific siRNA or with control siRNA for 48 h, washed and seeded alone or with GFP-CSCs. F-H. Downregulation by the indicated siRNA or control siRNA (48 h FAK and BMX, 72 h p130CAS) in ECs was confirmed by immunoblotting (F), then ECs were treated with p130CAS, FAK or BMX specific siRNA or with control siRNA, washed and seeded alone or with GFP-CSCs. I. ECs were treated with 1μM of the FAK or BMX inhibitor or DMSO (control), washed and seeded alone or with GFP-CSCs. Statistics: B-E, G-I, two-sided exact Wilcoxon rank-sum tests, and data graphed as the mean±SEM.
    Figure Legend Snippet: The direct interaction of ECs and CSCs promotes EC migration towards bFGF A. Diagrammatic representation of experimental design. Filters (3-μm pore) were coated on both sides with 20 μg/mL laminin. Red-fluorescent-ECs (3×10 4 ) were seeded alone or mixed with GFP-CSCs-(08387) (3×10 4 ) and co-seeded in growth factor-free NBM with 1% BSA on top of the filter. Growth factor-free NBM with 10 ng/ml bFGF was placed in the bottom chamber, and the cells allowed to migrate (37°C, 5% CO 2 ). At 6 h, cells were removed from the upper filter surface and cells on the lower filter surface were washed, fixed, photographed and quantified. B. The Src inhibitor PP2 (1 μM) was incubated with ECs (20 min), followed by washing, and EC seeding alone or co-seeding with CSCs. C. ECs were seeded in CM/EC+CSC (described in Figure 2B ), or co-seeded with CSCs. D, E. ECs were treated with integrin β3 specific siRNA, L1CAM specific siRNA or with control siRNA for 48 h, washed and seeded alone or with GFP-CSCs. F-H. Downregulation by the indicated siRNA or control siRNA (48 h FAK and BMX, 72 h p130CAS) in ECs was confirmed by immunoblotting (F), then ECs were treated with p130CAS, FAK or BMX specific siRNA or with control siRNA, washed and seeded alone or with GFP-CSCs. I. ECs were treated with 1μM of the FAK or BMX inhibitor or DMSO (control), washed and seeded alone or with GFP-CSCs. Statistics: B-E, G-I, two-sided exact Wilcoxon rank-sum tests, and data graphed as the mean±SEM.

    Techniques Used: Migration, Incubation

    65) Product Images from "Cooperation of Tyrosine Kinase Receptor TrkB and Epidermal Growth Factor Receptor Signaling Enhances Migration and Dispersal of Lung Tumor Cells"

    Article Title: Cooperation of Tyrosine Kinase Receptor TrkB and Epidermal Growth Factor Receptor Signaling Enhances Migration and Dispersal of Lung Tumor Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0100944

    TrkB expression induced cell spreading requires Akt and MEK kinase activity. Morphology of cell colonies and cell dispersal of A549 cells harbouring empty vector (e vector) or expressing wild-type TrkB (wt). Cells were plated on noncoated dishes, grown for 24 hours in the presence of 0.5% serum and subsequently stimulated with BDNF or EGF for a period of 24 hours in the presence or absence of Src inhibitor PP2, Akt kinase inhibitor Akti-1,2 (Akti) or MEK inhibitor PD0325901 (PD); untreated cells were cultured in the presence of vehicle (v, 0.1% DMSO). Bar, 200 µm. Bar graphs show quantification of colonies. Bars, mean ± SEM (n = 3).
    Figure Legend Snippet: TrkB expression induced cell spreading requires Akt and MEK kinase activity. Morphology of cell colonies and cell dispersal of A549 cells harbouring empty vector (e vector) or expressing wild-type TrkB (wt). Cells were plated on noncoated dishes, grown for 24 hours in the presence of 0.5% serum and subsequently stimulated with BDNF or EGF for a period of 24 hours in the presence or absence of Src inhibitor PP2, Akt kinase inhibitor Akti-1,2 (Akti) or MEK inhibitor PD0325901 (PD); untreated cells were cultured in the presence of vehicle (v, 0.1% DMSO). Bar, 200 µm. Bar graphs show quantification of colonies. Bars, mean ± SEM (n = 3).

    Techniques Used: Expressing, Activity Assay, Plasmid Preparation, Cell Culture

    66) Product Images from "Calcium Channels and Oxidative Stress Mediate a Synergistic Disruption of Tight Junctions by Ethanol and Acetaldehyde in Caco-2 Cell Monolayers"

    Article Title: Calcium Channels and Oxidative Stress Mediate a Synergistic Disruption of Tight Junctions by Ethanol and Acetaldehyde in Caco-2 Cell Monolayers

    Journal: Scientific Reports

    doi: 10.1038/srep38899

    Src kinase and MLCK activities mediate ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction. ( A–C ) Caco-2 cells were preincubated with PP2 (10 μM) or ML-7 (1 μM) 30 min prior to incubation with acetaldehyde (200 μM) in the presence of 75 mM ethanol. At 4-hour incubation, TER ( A ) and unidirectional flux of FITC-inulin ( B ) were measured. Fixed cell monolayers were stained for occludin (green) and ZO-1 (red) by immunofluorescence method ( C ). Values are mean ± SEM (n = 6). Asterisks indicate values that are significantly different from corresponding values for control cell monolayers (without ethanol). ( D ) Cell monolayers were preincubated with PP2 (10 μM) or ML-7 (1 μM) 30 min prior to incubation with acetaldehyde (400 μM). Inulin permeability was measured after 4-hour incubation. Values are mean ± SEM (n = 6). Asterisk indicates the value that is significantly (p
    Figure Legend Snippet: Src kinase and MLCK activities mediate ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction. ( A–C ) Caco-2 cells were preincubated with PP2 (10 μM) or ML-7 (1 μM) 30 min prior to incubation with acetaldehyde (200 μM) in the presence of 75 mM ethanol. At 4-hour incubation, TER ( A ) and unidirectional flux of FITC-inulin ( B ) were measured. Fixed cell monolayers were stained for occludin (green) and ZO-1 (red) by immunofluorescence method ( C ). Values are mean ± SEM (n = 6). Asterisks indicate values that are significantly different from corresponding values for control cell monolayers (without ethanol). ( D ) Cell monolayers were preincubated with PP2 (10 μM) or ML-7 (1 μM) 30 min prior to incubation with acetaldehyde (400 μM). Inulin permeability was measured after 4-hour incubation. Values are mean ± SEM (n = 6). Asterisk indicates the value that is significantly (p

    Techniques Used: Incubation, Staining, Immunofluorescence, Permeability

    67) Product Images from "In Vitro Enzymatic Characterization of Near Full Length EGFR in Activated and Inhibited States"

    Article Title: In Vitro Enzymatic Characterization of Near Full Length EGFR in Activated and Inhibited States

    Journal: Biochemistry

    doi: 10.1021/bi900755n

    tEGFR Autophosphorylation (A) Western blot analysis of the tEGFR:EGF complex before and after addition of ATP and in the presence and absence of the EGFR inhibitor erlotinib and the Src inhibitor PP2. The anti-phosphotyrosine antibody 4G10 was used for detection. The band near 50 kD is likely a C-terminal degradation product following cleavage in the extracellular region. (B) Western blot analysis of the tEGFR:EGF complex at various times (in minutes) following addition of ATP. A polyclonal antibody specific for EGFR Y845 (Life Sciences) was used.
    Figure Legend Snippet: tEGFR Autophosphorylation (A) Western blot analysis of the tEGFR:EGF complex before and after addition of ATP and in the presence and absence of the EGFR inhibitor erlotinib and the Src inhibitor PP2. The anti-phosphotyrosine antibody 4G10 was used for detection. The band near 50 kD is likely a C-terminal degradation product following cleavage in the extracellular region. (B) Western blot analysis of the tEGFR:EGF complex at various times (in minutes) following addition of ATP. A polyclonal antibody specific for EGFR Y845 (Life Sciences) was used.

    Techniques Used: Western Blot

    68) Product Images from "v-Src Causes Chromosome Bridges in a Caffeine-Sensitive Manner by Generating DNA Damage"

    Article Title: v-Src Causes Chromosome Bridges in a Caffeine-Sensitive Manner by Generating DNA Damage

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17060871

    v-Src induces DNA damage. ( A ) HCT116/v-Src cells were cultured with 1.0 μg/mL Dox for 12 h in the presence or absence of 10 μM PP2. The cells were fixed with PBS containing 4% paraformaldehyde and 20% methanol for 20 min at room temperature and stained for pTyr (green), γH2AX (red), and DNA (blue). Arrows indicate cells showing lower levels of pTyr staining. The boxed regions are magnified in the right panels. Bars, 20 μm; ( B ) HCT116/v-Src cells were cultured with or without 1.0 μg/mL Dox for 12 h. The cells were fixed with PBS containing 20% methanol and 4% formaldehyde for 20 min at room temperature and stained for γH2AX and DNA. The fluorescence signals were analyzed by an image analyzer. The left histogram shows the number of γH2AX foci per nucleus. The right histogram shows the total area of γH2AX foci per nucleus. The results represent the mean ± S.D. None, n = 2870; 1.0 μg/mL Dox, n = 686; ( C ) Cells were cultured with Dox at the indicated concentrations for three days or 300 ng/mL Adriamycin (ADR) for one day as a positive control. Whole cell lysates were subjected to a Western blot analysis. Blots were probed with anti-γH2AX and anti-actin (loading control). A representative result of two independent experiments is shown; ( D , E ) Cells were cultured with 1.0 μg/mL Dox for 12 h in the absence or presence of 10 μM PP2. Then, the cells were fixed and stained as described in A . The fluorescence signals were analyzed by an image analyzer. In D , fluorescence intensities of pTyr in individual cells were plotted with the mean ± S.D. ( n > 534) in the left, and data were converted to a histogram plot shown on the right; In E , fluorescence intensity of γH2AX in an individual nucleus was plotted with the mean ± S.D. ( n > 534) on the left, and data were converted to a histogram plot as shown on the right. p values were calculated using a two-tailed Student’s t -test.
    Figure Legend Snippet: v-Src induces DNA damage. ( A ) HCT116/v-Src cells were cultured with 1.0 μg/mL Dox for 12 h in the presence or absence of 10 μM PP2. The cells were fixed with PBS containing 4% paraformaldehyde and 20% methanol for 20 min at room temperature and stained for pTyr (green), γH2AX (red), and DNA (blue). Arrows indicate cells showing lower levels of pTyr staining. The boxed regions are magnified in the right panels. Bars, 20 μm; ( B ) HCT116/v-Src cells were cultured with or without 1.0 μg/mL Dox for 12 h. The cells were fixed with PBS containing 20% methanol and 4% formaldehyde for 20 min at room temperature and stained for γH2AX and DNA. The fluorescence signals were analyzed by an image analyzer. The left histogram shows the number of γH2AX foci per nucleus. The right histogram shows the total area of γH2AX foci per nucleus. The results represent the mean ± S.D. None, n = 2870; 1.0 μg/mL Dox, n = 686; ( C ) Cells were cultured with Dox at the indicated concentrations for three days or 300 ng/mL Adriamycin (ADR) for one day as a positive control. Whole cell lysates were subjected to a Western blot analysis. Blots were probed with anti-γH2AX and anti-actin (loading control). A representative result of two independent experiments is shown; ( D , E ) Cells were cultured with 1.0 μg/mL Dox for 12 h in the absence or presence of 10 μM PP2. Then, the cells were fixed and stained as described in A . The fluorescence signals were analyzed by an image analyzer. In D , fluorescence intensities of pTyr in individual cells were plotted with the mean ± S.D. ( n > 534) in the left, and data were converted to a histogram plot shown on the right; In E , fluorescence intensity of γH2AX in an individual nucleus was plotted with the mean ± S.D. ( n > 534) on the left, and data were converted to a histogram plot as shown on the right. p values were calculated using a two-tailed Student’s t -test.

    Techniques Used: Cell Culture, Staining, Fluorescence, Positive Control, Western Blot, Two Tailed Test

    69) Product Images from "Cooperation of p300 and PCAF in the Control of MicroRNA 200c/141 Transcription and Epithelial Characteristics"

    Article Title: Cooperation of p300 and PCAF in the Control of MicroRNA 200c/141 Transcription and Epithelial Characteristics

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0032449

    EMT induced by SPRR2a in HuCCT-1 involves loss of E-cadherin, increased vimentin, and reduction of miR-200 family transcription as compared to vector transfected controls. Examples of the morphological changes and changes in E-cadherin and vimentin expression in stable SPRR2a clones (NC = negative control) ( A ). Transcriptional loss of the miR200 family in SPRR2a expressing cells does not involve SH3 domain containing tyrosine kinases. Real-time PCR analysis of miR-200 family after 72 hrs treatment with ABL1 siRNA ( B ) and PP2 treatment ( C ) did not alter miR-200 expression. All clones used in this paper stably express SPRR2a ( D ). Real time PCR analysis: comparative 2-ΔΔCT method (miRNA: U6 internal control; ABL1: GAPDH internal control). (n = 2 independent experiments).
    Figure Legend Snippet: EMT induced by SPRR2a in HuCCT-1 involves loss of E-cadherin, increased vimentin, and reduction of miR-200 family transcription as compared to vector transfected controls. Examples of the morphological changes and changes in E-cadherin and vimentin expression in stable SPRR2a clones (NC = negative control) ( A ). Transcriptional loss of the miR200 family in SPRR2a expressing cells does not involve SH3 domain containing tyrosine kinases. Real-time PCR analysis of miR-200 family after 72 hrs treatment with ABL1 siRNA ( B ) and PP2 treatment ( C ) did not alter miR-200 expression. All clones used in this paper stably express SPRR2a ( D ). Real time PCR analysis: comparative 2-ΔΔCT method (miRNA: U6 internal control; ABL1: GAPDH internal control). (n = 2 independent experiments).

    Techniques Used: Plasmid Preparation, Transfection, Expressing, Clone Assay, Negative Control, Real-time Polymerase Chain Reaction, Stable Transfection

    70) Product Images from "NEDD9 and BCAR1 Negatively Regulate E-Cadherin Membrane Localization, and Promote E-Cadherin Degradation"

    Article Title: NEDD9 and BCAR1 Negatively Regulate E-Cadherin Membrane Localization, and Promote E-Cadherin Degradation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022102

    Treatment with SRC inhibitors reverses CAS-induced downregulation of E-cadherin. Immunofluorescence of MCF7 cells expressing vector pcDNA-GFP, pcDNA-GFP-NEDD9, pcDNA-GFP-BCAR1, or pcDNA-GFP-NEDD9 and pcDNA-GFP-BCAR1 (transfected cells shown in green) and treated with vehicle or PP2. Arrows indicate presence of E-cadherin (shown in red) at cell junctions in dasatinib or PP2-treated cells expressing vector, BCAR1 and/or NEDD9, but absence of E-cadherin in similarly transfected cells treated with vehicle. Scale bar, 20 µm.
    Figure Legend Snippet: Treatment with SRC inhibitors reverses CAS-induced downregulation of E-cadherin. Immunofluorescence of MCF7 cells expressing vector pcDNA-GFP, pcDNA-GFP-NEDD9, pcDNA-GFP-BCAR1, or pcDNA-GFP-NEDD9 and pcDNA-GFP-BCAR1 (transfected cells shown in green) and treated with vehicle or PP2. Arrows indicate presence of E-cadherin (shown in red) at cell junctions in dasatinib or PP2-treated cells expressing vector, BCAR1 and/or NEDD9, but absence of E-cadherin in similarly transfected cells treated with vehicle. Scale bar, 20 µm.

    Techniques Used: Immunofluorescence, Expressing, Plasmid Preparation, Transfection

    71) Product Images from "Cadherin complexes recruit mRNAs and RISC to regulate epithelial cell signaling"

    Article Title: Cadherin complexes recruit mRNAs and RISC to regulate epithelial cell signaling

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201612125

    Cadherin complexes recruit RISC to the apical junctions of polarized epithelial cells. Caco2 cells were grown to polarize and subjected to immunofluorescence for PLEKHA7 or p120 and the core proteins of the RISC complex Ago2 (A), GW182 (B), and PABPC1 (C). Images were obtained by confocal microscopy, and image stacks were acquired across the entire polarized monolayer; representative apical-basal images are shown. Enlarged parts of images on top of each stack indicate areas of cell–cell contact. (D) Western blots of PLEKHA7, p120, Ago2, GW182, and PABPC1 IPs of Caco2 cells for the same markers. IgG is the negative control. Molecular masses (kD) are indicated on the right. (E) Confocal microscopy images after immunofluorescence of Caco2 cells for GW182 and Ago2. Enlarged parts of images on top indicate areas of cell–cell contact. (F) Immunofluorescence of control (nontarget; NT) or PLEKHA7 knockdown (shPLEKHA7) Caco2 cells for Ago2, costained for PLEKHA7. PLEKHA7 background intracellular staining is an artifact of paraformaldehyde fixation. (G) Immunofluorescence of control (NT) or p120 knockdown (shp120) Caco2 cells for Ago2, costained for p120. (H) Immunofluorescence of control (NT) or E-cadherin knockdown (shE-cadherin) Caco2 cells for Ago2, costained for E-cadherin. (I) Caco2 cells treated with either vehicle control (DMSO) or the Src inhibitor PP2 were costained by immunofluorescence for p120 and Ago2. Colocalization of the Ago2 and p120 signals (bottom) was calculated using the Manders coefficient and expressed as fold change of the DMSO control (mean ± SD from n = 3 independent experiments; *, P
    Figure Legend Snippet: Cadherin complexes recruit RISC to the apical junctions of polarized epithelial cells. Caco2 cells were grown to polarize and subjected to immunofluorescence for PLEKHA7 or p120 and the core proteins of the RISC complex Ago2 (A), GW182 (B), and PABPC1 (C). Images were obtained by confocal microscopy, and image stacks were acquired across the entire polarized monolayer; representative apical-basal images are shown. Enlarged parts of images on top of each stack indicate areas of cell–cell contact. (D) Western blots of PLEKHA7, p120, Ago2, GW182, and PABPC1 IPs of Caco2 cells for the same markers. IgG is the negative control. Molecular masses (kD) are indicated on the right. (E) Confocal microscopy images after immunofluorescence of Caco2 cells for GW182 and Ago2. Enlarged parts of images on top indicate areas of cell–cell contact. (F) Immunofluorescence of control (nontarget; NT) or PLEKHA7 knockdown (shPLEKHA7) Caco2 cells for Ago2, costained for PLEKHA7. PLEKHA7 background intracellular staining is an artifact of paraformaldehyde fixation. (G) Immunofluorescence of control (NT) or p120 knockdown (shp120) Caco2 cells for Ago2, costained for p120. (H) Immunofluorescence of control (NT) or E-cadherin knockdown (shE-cadherin) Caco2 cells for Ago2, costained for E-cadherin. (I) Caco2 cells treated with either vehicle control (DMSO) or the Src inhibitor PP2 were costained by immunofluorescence for p120 and Ago2. Colocalization of the Ago2 and p120 signals (bottom) was calculated using the Manders coefficient and expressed as fold change of the DMSO control (mean ± SD from n = 3 independent experiments; *, P

    Techniques Used: Immunofluorescence, Confocal Microscopy, Western Blot, Negative Control, Staining

    72) Product Images from "Insulin-like Growth Factor (IGF) Signaling Requires ?v?3-IGF1-IGF Type 1 Receptor (IGF1R) Ternary Complex Formation in Anchorage Independence, and the Complex Formation Does Not Require IGF1R and Src Activation"

    Article Title: Insulin-like Growth Factor (IGF) Signaling Requires ?v?3-IGF1-IGF Type 1 Receptor (IGF1R) Ternary Complex Formation in Anchorage Independence, and the Complex Formation Does Not Require IGF1R and Src Activation

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.412536

    The effect of PPP (IGF1R kinase inhibitor) and PP2 (Src inhibitor) on IGF1 signaling in β3-CHO cells in anchorage-independent conditions. a , the effect of PPP and PP2 on cell survival. β3-CHO cells were plated in polyHEMA-coated plates,
    Figure Legend Snippet: The effect of PPP (IGF1R kinase inhibitor) and PP2 (Src inhibitor) on IGF1 signaling in β3-CHO cells in anchorage-independent conditions. a , the effect of PPP and PP2 on cell survival. β3-CHO cells were plated in polyHEMA-coated plates,

    Techniques Used:

    73) Product Images from "Positive Regulation of Lyn Kinase by CD148 Is Required for B Cell Receptor Signaling in B1 but Not B2 B Cells"

    Article Title: Positive Regulation of Lyn Kinase by CD148 Is Required for B Cell Receptor Signaling in B1 but Not B2 B Cells

    Journal: Immunity

    doi: 10.1016/j.immuni.2016.10.013

    Impaired SFK-Mediated BCR Signaling in CD148-Deficient B1 B Cells (A) Peritoneal B1 B cells (B220 lo CD23 − CD5 +/lo ) stimulated for 2 min with indicated concentrations of F(ab′) 2 anti-μ or 50 ng/mL phorbol myristate acetate (PMA) and stained with anti-phospho-Erk (pErk) antibody. (B) Peritoneal B1 B cells stimulated with 5 μg/mL of F(ab′) 2 anti-μ for the indicated times and stained as in (A). Shaded histograms indicate unstimulated cells. (C) Percentages of pErk + B1 B cells shown over time after stimulation with 5 μg/mL F(ab′) 2 anti-μ. (D) Percentages of pErk + follicular B cells (B220 + CD23 + CD21 − ) and MFI of pErk + MZ B cells (B220 + CD23 − CD21 + ) over time after stimulation with 10 μg/mL F(ab′) 2 anti-μ. (E) Total peritoneal lymphocytes or splenocytes loaded with Indo-1 and stained with anti-IgM Fab and dump stain to exclude non-B cells or peritoneal B2 B cells. Peritoneal cells and splenocytes were stimulated with the indicated concentrations of F(ab′) 2 anti-μ (closed arrow), followed by 1 nM ionomycin (open arrow). (F) Peritoneal lymphocytes stimulated for the indicated times with 1 μg/mL NP-18-ficoll. (G) Calcium mobilization of peritoneal B1 B cells stimulated with 5 μg/mL F(ab′) 2 anti-μ with or without 3 μM PP2. For (A) and (B), data are representative of seven individual experiments. For (C) and (D), n = 7 each of wild-type and Ptprj TM−/TM− mice. B1 and follicular B cells respond in a bimodal fashion, so percent pErk + .
    Figure Legend Snippet: Impaired SFK-Mediated BCR Signaling in CD148-Deficient B1 B Cells (A) Peritoneal B1 B cells (B220 lo CD23 − CD5 +/lo ) stimulated for 2 min with indicated concentrations of F(ab′) 2 anti-μ or 50 ng/mL phorbol myristate acetate (PMA) and stained with anti-phospho-Erk (pErk) antibody. (B) Peritoneal B1 B cells stimulated with 5 μg/mL of F(ab′) 2 anti-μ for the indicated times and stained as in (A). Shaded histograms indicate unstimulated cells. (C) Percentages of pErk + B1 B cells shown over time after stimulation with 5 μg/mL F(ab′) 2 anti-μ. (D) Percentages of pErk + follicular B cells (B220 + CD23 + CD21 − ) and MFI of pErk + MZ B cells (B220 + CD23 − CD21 + ) over time after stimulation with 10 μg/mL F(ab′) 2 anti-μ. (E) Total peritoneal lymphocytes or splenocytes loaded with Indo-1 and stained with anti-IgM Fab and dump stain to exclude non-B cells or peritoneal B2 B cells. Peritoneal cells and splenocytes were stimulated with the indicated concentrations of F(ab′) 2 anti-μ (closed arrow), followed by 1 nM ionomycin (open arrow). (F) Peritoneal lymphocytes stimulated for the indicated times with 1 μg/mL NP-18-ficoll. (G) Calcium mobilization of peritoneal B1 B cells stimulated with 5 μg/mL F(ab′) 2 anti-μ with or without 3 μM PP2. For (A) and (B), data are representative of seven individual experiments. For (C) and (D), n = 7 each of wild-type and Ptprj TM−/TM− mice. B1 and follicular B cells respond in a bimodal fashion, so percent pErk + .

    Techniques Used: Staining, Mouse Assay

    74) Product Images from "Mouse Mammary Tumor Virus Suppresses Apoptosis of Mammary Epithelial Cells through ITAM-Mediated Signaling"

    Article Title: Mouse Mammary Tumor Virus Suppresses Apoptosis of Mammary Epithelial Cells through ITAM-Mediated Signaling

    Journal: Journal of Virology

    doi: 10.1128/JVI.02029-12

    MMTV-HP expression suppresses apoptosis. (A) MMTV HP-, HP YY -, and BCR MAHB-transduced NMuMG cells were plated, and at 50% subconfluency, apoptosis was induced by serum starvation. PP2 was added to the HP- and MAHB-transduced cells at the beginning of
    Figure Legend Snippet: MMTV-HP expression suppresses apoptosis. (A) MMTV HP-, HP YY -, and BCR MAHB-transduced NMuMG cells were plated, and at 50% subconfluency, apoptosis was induced by serum starvation. PP2 was added to the HP- and MAHB-transduced cells at the beginning of

    Techniques Used: Expressing

    75) Product Images from "Endothelial Snail Regulates Capillary Branching Morphogenesis via Vascular Endothelial Growth Factor Receptor 3 Expression"

    Article Title: Endothelial Snail Regulates Capillary Branching Morphogenesis via Vascular Endothelial Growth Factor Receptor 3 Expression

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005324

    Snail is upregulated under extracellular matrix (ECM)-mediated signals. (A) Western blot analysis showing Snail expression on immobilized ECM. After HUVECs were transfected with siCon or siSnail, the transfectants were reseeded and cultured on PLL (20 μg/mL)-, FN (20 μg/mL)-, or CI (20 μg/mL)-coated culture dishes for 2 h. PLL, poly-L-lysine; FN, fibronectin; CI, collagen type I. (B) Time-course expression pattern of Snail on immobilized ECM. Confluent HUVECs were reseeded and cultured on PLL-, FN- or CI-coated dishes for the indicated time points. Snail expression was evaluated by western blot (upper) and quantitative RT-PCR (lower) analyses. (C) Western blot analysis showing the induction of phosphorylated Akt (p-Akt) and phosphorylated extracellular-regulated kinase 1/2 (p-Erk1/2) in HUVECs that were cultured on FN-coated dishes. (D) Snail expression on immobilized ECM after MK2206 treatment. Confluent HUVECs or human retinal endothelial cells (HRECs) were pre-exposed to 10 μM PP2 (a Src kinase inhibitor) or 1 μg/mL MK2206 (an allosteric Akt inhibitor) for 1 h, followed by reseeding and culture on PLL-, FN-, or CI-coated dishes for 2 h (western blot) or 1 h (quantitative RT-PCR).
    Figure Legend Snippet: Snail is upregulated under extracellular matrix (ECM)-mediated signals. (A) Western blot analysis showing Snail expression on immobilized ECM. After HUVECs were transfected with siCon or siSnail, the transfectants were reseeded and cultured on PLL (20 μg/mL)-, FN (20 μg/mL)-, or CI (20 μg/mL)-coated culture dishes for 2 h. PLL, poly-L-lysine; FN, fibronectin; CI, collagen type I. (B) Time-course expression pattern of Snail on immobilized ECM. Confluent HUVECs were reseeded and cultured on PLL-, FN- or CI-coated dishes for the indicated time points. Snail expression was evaluated by western blot (upper) and quantitative RT-PCR (lower) analyses. (C) Western blot analysis showing the induction of phosphorylated Akt (p-Akt) and phosphorylated extracellular-regulated kinase 1/2 (p-Erk1/2) in HUVECs that were cultured on FN-coated dishes. (D) Snail expression on immobilized ECM after MK2206 treatment. Confluent HUVECs or human retinal endothelial cells (HRECs) were pre-exposed to 10 μM PP2 (a Src kinase inhibitor) or 1 μg/mL MK2206 (an allosteric Akt inhibitor) for 1 h, followed by reseeding and culture on PLL-, FN-, or CI-coated dishes for 2 h (western blot) or 1 h (quantitative RT-PCR).

    Techniques Used: Western Blot, Expressing, Transfection, Cell Culture, Quantitative RT-PCR

    Related Articles

    Functional Assay:

    Article Title: HMBPP analog prodrugs bypass energy-dependent uptake to promote efficient BTN3A1-mediated malignant cell lysis by Vγ9Vδ2 T lymphocyte effectors
    Article Snippet: .. PE-BTN3A1 (CD277) antibody (BT3.1), FITC-CD183 (G025H7), functional grade anti-human TCR γ/δ Antibody (B1), and human IFNγ ELISA MAX deluxe kit were obtained from Biolegend while G418, PP2 and phytohaemagglutinin P were obtained from Sigma. .. Anti-myc (9E10) antibody was purified from hybridoma cells obtained from the Developmental Studies Hybridoma Bank at The University of Iowa.

    Luciferase:

    Article Title: Angiomotin-like2 Gene (amotl2) Is Required for Migration and Proliferation of Endothelial Cells during Angiogenesis *
    Article Snippet: Paragraph title: Cell Culture, Luciferase Assay, and Immunoblotting ... For chemical treatments, cells were serum-deprived for 2 h and then treated with 20 μ m PP2 (Src inhibitor from Calbiochem) or 10 μ m U5402 (FGFR1 inhibitor from Calbiochem) for 2 h at 37 °C prior to FGF2 stimulation.

    Positive Control:

    Article Title: Semaphorin 4D/Plexin-B1 Induces Endothelial Cell Migration through the Activation of PYK2, Src, and the Phosphatidylinositol 3-Kinase-Akt Pathway
    Article Snippet: Ten percent fetal bovine serum was used as a positive control, and serum-free HAM F-12 containing 0.1% bovine serum albumin (BSA) was the negative control. .. Cells were also treated with 10 μM PP2 (Calbiochem, La Jolla, CA), 50 μM (Biosource), or vehicle control prior to migration.

    Enzyme-linked Immunosorbent Assay:

    Article Title: HMBPP analog prodrugs bypass energy-dependent uptake to promote efficient BTN3A1-mediated malignant cell lysis by Vγ9Vδ2 T lymphocyte effectors
    Article Snippet: .. PE-BTN3A1 (CD277) antibody (BT3.1), FITC-CD183 (G025H7), functional grade anti-human TCR γ/δ Antibody (B1), and human IFNγ ELISA MAX deluxe kit were obtained from Biolegend while G418, PP2 and phytohaemagglutinin P were obtained from Sigma. .. Anti-myc (9E10) antibody was purified from hybridoma cells obtained from the Developmental Studies Hybridoma Bank at The University of Iowa.

    Incubation:

    Article Title: Phospholipase C?2 is required for basal but not oestrogen deficiency-induced bone resorption
    Article Snippet: .. For signalling studies, macrophages were cultured for 5–8 days in bacterial dishes, suspended with 5 mM EDTA and serum starved for 6 h. When indicated, the cells were then incubated with 10 μM PP2 (EMD Biosciences, Darmstadt, Germany) for 8 min. ..

    Immunocytochemistry:

    Article Title: Identification of a functional interaction between Kv4.3 channels and c-Src tyrosine kinase
    Article Snippet: Secondary antibodies for Western blot were Alexa Fluor 680 anti-rabbit (Invitrogen, Carlsbad, CA) and IRDye 800 anti-mouse (Rockland Immunochemicals, Gilbertsville, PA) and immunocytochemistry were rhodamine-conjugated donkey anti-rabbit IgG and FITC-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). .. PP2, PP3 and bpV(phen) were obtained from Calbiochem (San Diego, CA).

    Activity Assay:

    Article Title: Angiomotin-like2 Gene (amotl2) Is Required for Migration and Proliferation of Endothelial Cells during Angiogenesis *
    Article Snippet: At 48 h after transfection with the ERK reporter ELK-luciferase, cells were harvested with passive lysis buffer (Promega, Madison, WI), and luciferase activity was then measured using a luminometer (Berthold Technologies, Oak Ridge, TN). .. For chemical treatments, cells were serum-deprived for 2 h and then treated with 20 μ m PP2 (Src inhibitor from Calbiochem) or 10 μ m U5402 (FGFR1 inhibitor from Calbiochem) for 2 h at 37 °C prior to FGF2 stimulation.

    Cell Culture:

    Article Title: Phospholipase C?2 is required for basal but not oestrogen deficiency-induced bone resorption
    Article Snippet: .. For signalling studies, macrophages were cultured for 5–8 days in bacterial dishes, suspended with 5 mM EDTA and serum starved for 6 h. When indicated, the cells were then incubated with 10 μM PP2 (EMD Biosciences, Darmstadt, Germany) for 8 min. ..

    Article Title: Angiomotin-like2 Gene (amotl2) Is Required for Migration and Proliferation of Endothelial Cells during Angiogenesis *
    Article Snippet: Paragraph title: Cell Culture, Luciferase Assay, and Immunoblotting ... For chemical treatments, cells were serum-deprived for 2 h and then treated with 20 μ m PP2 (Src inhibitor from Calbiochem) or 10 μ m U5402 (FGFR1 inhibitor from Calbiochem) for 2 h at 37 °C prior to FGF2 stimulation.

    Article Title: UVB-mediated down-regulation of proteasome in cultured human primary pterygium fibroblasts
    Article Snippet: .. After each exposure, cells were placed in fresh serum-free DMEM-0.2% LH and cultured for additional 24 h. When the Oltipraz (Sigma-Aldrich Chemical Co., St Louis, MO) or PP2 (Sigma-Aldrich Chemical Co., St Louis, MO) were used, the cells were pre-incubated in the presence of respective factor in serum-free DMEM-0.2% LH, for 2 h before the irradiation. ..

    Article Title: Cyclic stretch disrupts apical junctional complexes in Caco-2 cell monolayers by a JNK-2-, c-Src-, and MLCK-dependent mechanism
    Article Snippet: Cell culture medium (DMEM), FBS, and antibiotics were procured from Cellgrow (Manassas, VA). .. SP600125 (JNK inhibitor), ML-7 (MLCK inhibitor), and PP2 (Src kinase inhibitor) were purchased from EMD Chemicals (San Diego, CA).

    Expressing:

    Article Title: Phospholipase C?2 is required for basal but not oestrogen deficiency-induced bone resorption
    Article Snippet: Biochemical and signalling studies PLCγ2 expression was determined from Triton X-100-soluble whole osteoclast or macrophage lysates as described [ ]. .. For signalling studies, macrophages were cultured for 5–8 days in bacterial dishes, suspended with 5 mM EDTA and serum starved for 6 h. When indicated, the cells were then incubated with 10 μM PP2 (EMD Biosciences, Darmstadt, Germany) for 8 min.

    Western Blot:

    Article Title: Identification of a functional interaction between Kv4.3 channels and c-Src tyrosine kinase
    Article Snippet: Secondary antibodies for Western blot were Alexa Fluor 680 anti-rabbit (Invitrogen, Carlsbad, CA) and IRDye 800 anti-mouse (Rockland Immunochemicals, Gilbertsville, PA) and immunocytochemistry were rhodamine-conjugated donkey anti-rabbit IgG and FITC-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). .. PP2, PP3 and bpV(phen) were obtained from Calbiochem (San Diego, CA).

    Immunohistochemistry:

    Article Title: RANKL/RANK pathway abrogates cetuximab sensitivity in gastric cancer cells via activation of EGFR and c-Src
    Article Snippet: PP2 was obtained from Sigma-Aldrich Co. (St Louis, MO, USA). .. S-P immunohistochemical kit and 3,3′-diaminobenzidine tetrahydrochloride kit were obtained from Maixin Bio (Fuzhou Maixin Biological Technology Ltd., Fujian, People’s Republic of China).

    Infection:

    Article Title: Express Path Analysis Identifies a Tyrosine Kinase Src-centric Network Regulating Divergent Host Responses to Mycobacterium tuberculosis Infection *
    Article Snippet: .. The cells were then maintained in complete media containing PP2 inhibitor at a concentration of 50 nm for 48 h. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) at 48 h post-bacterial infection. .. The fixed cells were stained with DAPI (300 nm in 1× PBS).

    other:

    Article Title: Metalloproteinase/Presenilin1 processing of ephrinB regulates EphB-induced Src phosphorylation and signaling
    Article Snippet: L-685,458, lactacystin, compound E and Src kinase inhibitors SU6656 and PP2 were from Calbiochem; GM6001 from Chemicon; TPA, fibrinogen, thrombin, aprotinin, microcarrier (MC) beads (Cytodex-3) and Hoechst 33258 from Sigma.

    Article Title: Rapamycin Induces Transactivation of the EGFR and Increases Cell Survival
    Article Snippet: Rapamycin, AG1478, TAPI-2, GM6001 and PP2/PP3 were from Calbiochem Inc. (San Diego, CA).

    Article Title: Epidermal Growth Factor Receptor Transactivation Is Required for Mitogen-Activated Protein Kinase Activation by Muscarinic Acetylcholine Receptors in HaCaT Keratinocytes
    Article Snippet: Reagents Human EGF, CCh, nicotine, atropine, telenzepine, 4-DAMP, tropicamide, PP2 and Ly 294002 were purchased from Sigma-Aldrich (Taufkirchen, Germany).

    Injection:

    Article Title: Integrin α6β4-Src-AKT signaling induces cellular senescence by counteracting apoptosis in irradiated tumor cells and tissues
    Article Snippet: .. When the tumor reached an average volume of ~100 mm3 , an integrin β4 SiRNA (Bioneer, Daejeon, Korea) mixture or PP2 (Calbiochem) with AteloGenes Local Use (KOKEN, Koraku, Tokyo) was injected to surround the whole tumor mass. ..

    Recombinant:

    Article Title: RANKL/RANK pathway abrogates cetuximab sensitivity in gastric cancer cells via activation of EGFR and c-Src
    Article Snippet: Recombinant sRANKL and rOPG was purchased from CytoLab/PeproTech Asia (USA). .. PP2 was obtained from Sigma-Aldrich Co. (St Louis, MO, USA).

    Article Title: Identification of a functional interaction between Kv4.3 channels and c-Src tyrosine kinase
    Article Snippet: PP2, PP3 and bpV(phen) were obtained from Calbiochem (San Diego, CA). .. Purified recombinant c-Src kinase was from Upstate.

    Immunofluorescence:

    Article Title: The Focal Adhesion: A Regulated Component of Aortic Stiffness
    Article Snippet: PP2 was purchased from EMD Biosciences (La Jolla, CA); FAK inhibitor 14 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA); and ML-9 was purchased from Calbiochem (La Jolla, CA). .. For immunofluorescence experiments, goat anti-rabbit and goat anti-mouse Alexa Fluor 488 and Alexa Fluor 568 (1∶1,000; Invitrogen) were used as secondary antibodies.

    Magnetic Cell Separation:

    Article Title: HMBPP analog prodrugs bypass energy-dependent uptake to promote efficient BTN3A1-mediated malignant cell lysis by Vγ9Vδ2 T lymphocyte effectors
    Article Snippet: IL-2 and the MACS γδ T cell negative selection kit were obtained through Miltenyi. .. PE-BTN3A1 (CD277) antibody (BT3.1), FITC-CD183 (G025H7), functional grade anti-human TCR γ/δ Antibody (B1), and human IFNγ ELISA MAX deluxe kit were obtained from Biolegend while G418, PP2 and phytohaemagglutinin P were obtained from Sigma.

    In Vivo:

    Article Title: Integrin α6β4-Src-AKT signaling induces cellular senescence by counteracting apoptosis in irradiated tumor cells and tissues
    Article Snippet: Paragraph title: Tumor-xenograft mice and in vivo experiments ... When the tumor reached an average volume of ~100 mm3 , an integrin β4 SiRNA (Bioneer, Daejeon, Korea) mixture or PP2 (Calbiochem) with AteloGenes Local Use (KOKEN, Koraku, Tokyo) was injected to surround the whole tumor mass.

    Fluorescence:

    Article Title: Semaphorin 4D/Plexin-B1 Induces Endothelial Cell Migration through the Activation of PYK2, Src, and the Phosphatidylinositol 3-Kinase-Akt Pathway
    Article Snippet: Where indicated, cells were transfected with pCEFL control or pCEFL DN-PYK2 using Lipofectamine Plus (Invitrogen) supplemented with CombiMag transfection agent (Oz Biosciences, Marseille, France) to achieve high transfection efficiency (determined to be ≥76% by fluorescence-activated cell sorter analysis of pCEFL enhanced green fluorescent protein [EGFP]-transfected endothelial cells). .. Cells were also treated with 10 μM PP2 (Calbiochem, La Jolla, CA), 50 μM (Biosource), or vehicle control prior to migration.

    Transfection:

    Article Title: Angiomotin-like2 Gene (amotl2) Is Required for Migration and Proliferation of Endothelial Cells during Angiogenesis *
    Article Snippet: Transfection efficiency was normalized by Renilla . .. For chemical treatments, cells were serum-deprived for 2 h and then treated with 20 μ m PP2 (Src inhibitor from Calbiochem) or 10 μ m U5402 (FGFR1 inhibitor from Calbiochem) for 2 h at 37 °C prior to FGF2 stimulation.

    Article Title: Cyclic stretch disrupts apical junctional complexes in Caco-2 cell monolayers by a JNK-2-, c-Src-, and MLCK-dependent mechanism
    Article Snippet: Transfection reagents, Opti-MEM, Oligofectamine, and Plus were purchased from Invitrogen (Carlsbad, CA). .. SP600125 (JNK inhibitor), ML-7 (MLCK inhibitor), and PP2 (Src kinase inhibitor) were purchased from EMD Chemicals (San Diego, CA).

    Article Title: Semaphorin 4D/Plexin-B1 Induces Endothelial Cell Migration through the Activation of PYK2, Src, and the Phosphatidylinositol 3-Kinase-Akt Pathway
    Article Snippet: Where indicated, cells were transfected with pCEFL control or pCEFL DN-PYK2 using Lipofectamine Plus (Invitrogen) supplemented with CombiMag transfection agent (Oz Biosciences, Marseille, France) to achieve high transfection efficiency (determined to be ≥76% by fluorescence-activated cell sorter analysis of pCEFL enhanced green fluorescent protein [EGFP]-transfected endothelial cells). .. Cells were also treated with 10 μM PP2 (Calbiochem, La Jolla, CA), 50 μM (Biosource), or vehicle control prior to migration.

    Microscopy:

    Article Title: Express Path Analysis Identifies a Tyrosine Kinase Src-centric Network Regulating Divergent Host Responses to Mycobacterium tuberculosis Infection *
    Article Snippet: The cells were then maintained in complete media containing PP2 inhibitor at a concentration of 50 nm for 48 h. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) at 48 h post-bacterial infection. .. Four fields were acquired randomly from each set of stained cells with a Nikon EclipseTi-E laser-scanning confocal microscope equipped with a 20×/0.75 numerical aperture plan apochromat differential interference contrast objective lens using the blue diode laser (excitation at 408 nm and emission at 450 nm ) and argon laser (excitation at 488 nm and emission at 515 nm).

    Purification:

    Article Title: HMBPP analog prodrugs bypass energy-dependent uptake to promote efficient BTN3A1-mediated malignant cell lysis by Vγ9Vδ2 T lymphocyte effectors
    Article Snippet: PE-BTN3A1 (CD277) antibody (BT3.1), FITC-CD183 (G025H7), functional grade anti-human TCR γ/δ Antibody (B1), and human IFNγ ELISA MAX deluxe kit were obtained from Biolegend while G418, PP2 and phytohaemagglutinin P were obtained from Sigma. .. Anti-myc (9E10) antibody was purified from hybridoma cells obtained from the Developmental Studies Hybridoma Bank at The University of Iowa.

    Article Title: Identification of a functional interaction between Kv4.3 channels and c-Src tyrosine kinase
    Article Snippet: PP2, PP3 and bpV(phen) were obtained from Calbiochem (San Diego, CA). .. Purified recombinant c-Src kinase was from Upstate.

    Diff-Quik:

    Article Title: Semaphorin 4D/Plexin-B1 Induces Endothelial Cell Migration through the Activation of PYK2, Src, and the Phosphatidylinositol 3-Kinase-Akt Pathway
    Article Snippet: After 7 h, the chamber was disassembled and the membrane was stained with Diff-Quick Stain (Dade Behring, Deerfield, Illinois), placed on a glass slide, and scanned. .. Cells were also treated with 10 μM PP2 (Calbiochem, La Jolla, CA), 50 μM (Biosource), or vehicle control prior to migration.

    Confocal Microscopy:

    Article Title: Express Path Analysis Identifies a Tyrosine Kinase Src-centric Network Regulating Divergent Host Responses to Mycobacterium tuberculosis Infection *
    Article Snippet: Paragraph title: Confocal Microscopy ... The cells were then maintained in complete media containing PP2 inhibitor at a concentration of 50 nm for 48 h. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) at 48 h post-bacterial infection.

    CRISPR:

    Article Title: HMBPP analog prodrugs bypass energy-dependent uptake to promote efficient BTN3A1-mediated malignant cell lysis by Vγ9Vδ2 T lymphocyte effectors
    Article Snippet: DiD, calcein AM, pcDNA3.1 and DNA primers were obtained through Life Technologies, while CRISPR/Cas9 repair templates were obtained through IDT. .. PE-BTN3A1 (CD277) antibody (BT3.1), FITC-CD183 (G025H7), functional grade anti-human TCR γ/δ Antibody (B1), and human IFNγ ELISA MAX deluxe kit were obtained from Biolegend while G418, PP2 and phytohaemagglutinin P were obtained from Sigma.

    Mouse Assay:

    Article Title: Integrin α6β4-Src-AKT signaling induces cellular senescence by counteracting apoptosis in irradiated tumor cells and tissues
    Article Snippet: Paragraph title: Tumor-xenograft mice and in vivo experiments ... When the tumor reached an average volume of ~100 mm3 , an integrin β4 SiRNA (Bioneer, Daejeon, Korea) mixture or PP2 (Calbiochem) with AteloGenes Local Use (KOKEN, Koraku, Tokyo) was injected to surround the whole tumor mass.

    Software:

    Article Title: Semaphorin 4D/Plexin-B1 Induces Endothelial Cell Migration through the Activation of PYK2, Src, and the Phosphatidylinositol 3-Kinase-Akt Pathway
    Article Snippet: Densitometric quantitation was performed with National Institutes of Health image software, and cell migration was expressed as staining intensity relative to the negative control wells. .. Cells were also treated with 10 μM PP2 (Calbiochem, La Jolla, CA), 50 μM (Biosource), or vehicle control prior to migration.

    Irradiation:

    Article Title: Integrin α6β4-Src-AKT signaling induces cellular senescence by counteracting apoptosis in irradiated tumor cells and tissues
    Article Snippet: When the tumor reached an average volume of ~100 mm3 , an integrin β4 SiRNA (Bioneer, Daejeon, Korea) mixture or PP2 (Calbiochem) with AteloGenes Local Use (KOKEN, Koraku, Tokyo) was injected to surround the whole tumor mass. .. Local regional irradiation of xenografted tumor was performed under anesthesia; irradiation was achieved using a 60 Co source irradiator (Theratron 780; Atomic Energy of Canada Ltd.) operating at 1.3 Gy/min.

    Article Title: UVB-mediated down-regulation of proteasome in cultured human primary pterygium fibroblasts
    Article Snippet: .. After each exposure, cells were placed in fresh serum-free DMEM-0.2% LH and cultured for additional 24 h. When the Oltipraz (Sigma-Aldrich Chemical Co., St Louis, MO) or PP2 (Sigma-Aldrich Chemical Co., St Louis, MO) were used, the cells were pre-incubated in the presence of respective factor in serum-free DMEM-0.2% LH, for 2 h before the irradiation. ..

    Negative Control:

    Article Title: Semaphorin 4D/Plexin-B1 Induces Endothelial Cell Migration through the Activation of PYK2, Src, and the Phosphatidylinositol 3-Kinase-Akt Pathway
    Article Snippet: Densitometric quantitation was performed with National Institutes of Health image software, and cell migration was expressed as staining intensity relative to the negative control wells. .. Cells were also treated with 10 μM PP2 (Calbiochem, La Jolla, CA), 50 μM (Biosource), or vehicle control prior to migration.

    Selection:

    Article Title: HMBPP analog prodrugs bypass energy-dependent uptake to promote efficient BTN3A1-mediated malignant cell lysis by Vγ9Vδ2 T lymphocyte effectors
    Article Snippet: IL-2 and the MACS γδ T cell negative selection kit were obtained through Miltenyi. .. PE-BTN3A1 (CD277) antibody (BT3.1), FITC-CD183 (G025H7), functional grade anti-human TCR γ/δ Antibody (B1), and human IFNγ ELISA MAX deluxe kit were obtained from Biolegend while G418, PP2 and phytohaemagglutinin P were obtained from Sigma.

    Size-exclusion Chromatography:

    Article Title: UVB-mediated down-regulation of proteasome in cultured human primary pterygium fibroblasts
    Article Snippet: The exposure time (t) (in sec) was determined by the equation t = dose (mJ/cm2 )/fluence rate (mW/cm2 ) [ ]. .. After each exposure, cells were placed in fresh serum-free DMEM-0.2% LH and cultured for additional 24 h. When the Oltipraz (Sigma-Aldrich Chemical Co., St Louis, MO) or PP2 (Sigma-Aldrich Chemical Co., St Louis, MO) were used, the cells were pre-incubated in the presence of respective factor in serum-free DMEM-0.2% LH, for 2 h before the irradiation.

    Quantitation Assay:

    Article Title: Semaphorin 4D/Plexin-B1 Induces Endothelial Cell Migration through the Activation of PYK2, Src, and the Phosphatidylinositol 3-Kinase-Akt Pathway
    Article Snippet: Densitometric quantitation was performed with National Institutes of Health image software, and cell migration was expressed as staining intensity relative to the negative control wells. .. Cells were also treated with 10 μM PP2 (Calbiochem, La Jolla, CA), 50 μM (Biosource), or vehicle control prior to migration.

    Concentration Assay:

    Article Title: Express Path Analysis Identifies a Tyrosine Kinase Src-centric Network Regulating Divergent Host Responses to Mycobacterium tuberculosis Infection *
    Article Snippet: .. The cells were then maintained in complete media containing PP2 inhibitor at a concentration of 50 nm for 48 h. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) at 48 h post-bacterial infection. .. The fixed cells were stained with DAPI (300 nm in 1× PBS).

    Article Title: An RPTPα/Src family kinase/Rap1 signaling module recruits myosin IIB to support contractile tension at apical E-cadherin junctions
    Article Snippet: .. Cells were treated for 1 h with PP2 (529573; Calbiochem) or Y-27632 (688000; Merck) at a final concentration of 25 and 30 μM, respectively. .. Immunofluorescence imaging Cells were fixed with 10% trichloroacetic acid on ice for 15 min for RPTPσ staining or fixed in 4% paraformaldehyde in cytoskeletal stabilization buffer (10 mM PIPES pH 6.8, 100 mM KCl, 300 mM sucrose, 2 mM EGTA, 2 mM MgCl2 ) on ice for 30 min and subsequently treated with 50 mM NH4 Cl for 10 min. Permeabilization was then performed with 0.2% saponin/0.1% bovine serum albumin in Tris-buffered saline.

    Migration:

    Article Title: Semaphorin 4D/Plexin-B1 Induces Endothelial Cell Migration through the Activation of PYK2, Src, and the Phosphatidylinositol 3-Kinase-Akt Pathway
    Article Snippet: .. Cells were also treated with 10 μM PP2 (Calbiochem, La Jolla, CA), 50 μM (Biosource), or vehicle control prior to migration. .. The chemoattractants used were 200 ng/ml soluble Semaphorin 4D or 100 ng/ml NGF (Upstate Biotechnology).

    Lysis:

    Article Title: Angiomotin-like2 Gene (amotl2) Is Required for Migration and Proliferation of Endothelial Cells during Angiogenesis *
    Article Snippet: At 48 h after transfection with the ERK reporter ELK-luciferase, cells were harvested with passive lysis buffer (Promega, Madison, WI), and luciferase activity was then measured using a luminometer (Berthold Technologies, Oak Ridge, TN). .. For chemical treatments, cells were serum-deprived for 2 h and then treated with 20 μ m PP2 (Src inhibitor from Calbiochem) or 10 μ m U5402 (FGFR1 inhibitor from Calbiochem) for 2 h at 37 °C prior to FGF2 stimulation.

    Staining:

    Article Title: Semaphorin 4D/Plexin-B1 Induces Endothelial Cell Migration through the Activation of PYK2, Src, and the Phosphatidylinositol 3-Kinase-Akt Pathway
    Article Snippet: Densitometric quantitation was performed with National Institutes of Health image software, and cell migration was expressed as staining intensity relative to the negative control wells. .. Cells were also treated with 10 μM PP2 (Calbiochem, La Jolla, CA), 50 μM (Biosource), or vehicle control prior to migration.

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  • 74
    Millipore fyn inhibitor pp2
    <t>Fyn</t> expression and activation in the hypothalmus. (A–B) Some rats were used without previous treatment (A), or some rats were treated with Fyn inhitor <t>PP2</t> (5 nmol in 100 µl buffer, ip) 30 min before leptin injection (B). Anesthetized rats were injected via intra cava vein either with 100 µl saline (C) or with an equal volume of leptin (10 −6 M) (Lep and PP2+Lep); the hypothalami were obtained, homogenized and samples containing 0.5 mg total protein were used in immunoprecipitation assays with anti-Fyn or anti-JAK2 antibodies; immunocomplexes were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-phosphotyrosine antibody; or 0.2 mg protein was separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-Fyn or anti-JAK2 antibodies. The depicted blots are representative of n = 5. In all experiments, n = 5; *p
    Fyn Inhibitor Pp2, supplied by Millipore, used in various techniques. Bioz Stars score: 74/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore src inhibitor pp2
    VEGF induced-VE-cadherin extracellular domain cleavage is preceded by a <t>Src-dependent</t> VE-cadherin tyrosine phosphorylation. (A,B) HUVECs treated with VEGF (50 ng/mL) were analyzed for phosphotyrosinated-VE-cadherin in cell extracts (A) and VE-cadherin extracellular domain in conditioned medium (B): (A) VE-cadherin was immunoprecipitated from 200 µg of protein lysates and analyzed by SDS-PAGE and western blotting with the anti-phosphotyrosine antibody. VEGF induced a time-dependent tyrosine phosphorylation of VE-cadherin detectable after 2 min of stimulation. (B) Conditioned media from VEGF-stimulated HUVECs were concentrated and analyzed by SDS-PAGE and western blotting with human VE-cadherin antibody directed against VE-cadherin extracellular domain (BV9). A 90 kDa fragment corresponding to the full length VE-cadherin extracellular domain was already detectable after 10 min of VEGF stimulation. (C) HUVECs were pretreated with increasing concentrations of Src inhibitor <t>PP2</t> (2.5 to 20 µM) for 15 min, prior to treatment with VEGF for 15 min. VE-cadherin was immunoprecipitated from 200 µg of protein lysates. PP2 concentrations higher than 2.5 µM completely inhibited VEGF-induced VE-cadherin tyrosine phosphorylation. (D) Analysis of conditioned media from an identical number of HUVECs pre-treated for 15 min with PP2 (5 µM) before VEGF stimulation for 15 min: the inhibitor decreased VEGF-induced VE-cadherin cleavage. (E,F) Src expression was inhibited by Src-siRNA in HUVECs 24 hours before VEGF stimulation (15min). Analysis of conditioned media showed that the knock-down of Src (controlled in F) decreased the level of soluble VE-cadherin in the media. (D,E,F) The signals were quantified using ImageJ software, error bars in graphs indicate S.D. and experiments were repeated at least three times in a similar configuration.
    Src Inhibitor Pp2, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/src inhibitor pp2/product/Millipore
    Average 97 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    src inhibitor pp2 - by Bioz Stars, 2020-02
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    97
    Millipore sfk inhibitor pp2
    Inhibition of Src family kinase activity blocks tyrosine phosphorylation of SLC11A1. U937-SLC11A1 cells were cultured with PMA (10ng/ml) for 48 hrs and were then left untreated (CTR) or treated with <t>PP2</t> or PP3 (inactive analogue) for another 24 hours. Cell lysates were prepared. (A) c-Src and active c-Src (pY418) were monitored by Western blotting analysis. (B) Cell lysates were immunoprecipitated with the anti-c-Myc antibody 9E10, and the immunoprecipitates were probed with antibody 4G10 to phosphotyrosine for phosphorylated SLC11A1. The Western blots were stripped and re-probed with an antibody against SLC11A1. (C) The level of SLC11A1 phosphorylation was quantified by densitometry analysis and normalized to the level of total SLC11A1 protein. The relative phosphorylation level of SLC11A1 in untreated control group was set as 100%. The inhibitory effect of PP2 on SLC11A1 phosphorylation was assessed in 3 separate experiments (mean± SE). *** P
    Sfk Inhibitor Pp2, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sfk inhibitor pp2/product/Millipore
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    Image Search Results


    Fyn expression and activation in the hypothalmus. (A–B) Some rats were used without previous treatment (A), or some rats were treated with Fyn inhitor PP2 (5 nmol in 100 µl buffer, ip) 30 min before leptin injection (B). Anesthetized rats were injected via intra cava vein either with 100 µl saline (C) or with an equal volume of leptin (10 −6 M) (Lep and PP2+Lep); the hypothalami were obtained, homogenized and samples containing 0.5 mg total protein were used in immunoprecipitation assays with anti-Fyn or anti-JAK2 antibodies; immunocomplexes were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-phosphotyrosine antibody; or 0.2 mg protein was separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-Fyn or anti-JAK2 antibodies. The depicted blots are representative of n = 5. In all experiments, n = 5; *p

    Journal: PLoS ONE

    Article Title: Fyn Mediates Leptin Actions in the Thymus of Rodents

    doi: 10.1371/journal.pone.0007707

    Figure Lengend Snippet: Fyn expression and activation in the hypothalmus. (A–B) Some rats were used without previous treatment (A), or some rats were treated with Fyn inhitor PP2 (5 nmol in 100 µl buffer, ip) 30 min before leptin injection (B). Anesthetized rats were injected via intra cava vein either with 100 µl saline (C) or with an equal volume of leptin (10 −6 M) (Lep and PP2+Lep); the hypothalami were obtained, homogenized and samples containing 0.5 mg total protein were used in immunoprecipitation assays with anti-Fyn or anti-JAK2 antibodies; immunocomplexes were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-phosphotyrosine antibody; or 0.2 mg protein was separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-Fyn or anti-JAK2 antibodies. The depicted blots are representative of n = 5. In all experiments, n = 5; *p

    Article Snippet: Leptin, JAK inhibitor AG490 (tyrphostin B42) and Fyn inhibitor PP2 were from Calbiochem (La Jolla, CA, USA).

    Techniques: Expressing, Activation Assay, Injection, Immunoprecipitation, SDS Page

    Effects of Fyn inhibition on apoptosis and cytokine expression. (A) Rats were treated ip for three days with Fyn antisense phosphothioate modified oligonucleotide (FynAS) (400 µl, 2 nmol). On the fourth day, the rats were injected via intra cava vein either with 100 µl saline (C and FynAS) or with an equal volume of leptin (10 −6 M) (Lep and FynAS+Lep); the thymuses were obtained, homogenized and 0.2 mg protein was separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-Bax or anti-Bcl-2 antibodies. (B) Isolated thymocytes were treated with leptin (10 −8 M)(Lep) or PP2 (10 −8 M) or both together and apoptosis was determined by the annexin method after 24h. (C–D) Rats were treated ip with a single dose of 100 µL of saline solution (C); 100 µL of lipopolysaccharide (LPS) 1 mg/mL; 150 µL of PP2 5 nM; 100 µL of leptin 31.2 µM (Lep) or with different combinations of these treatments; the sequence of treatment was, PP2, followed by leptin after 30 min and LPS after 30 min. Thymus was obtained after 2 h and RNA was prepared for determination of IL-1b (C) and TNF-α expression by real-time PCR. In all experiments n = 5. *p

    Journal: PLoS ONE

    Article Title: Fyn Mediates Leptin Actions in the Thymus of Rodents

    doi: 10.1371/journal.pone.0007707

    Figure Lengend Snippet: Effects of Fyn inhibition on apoptosis and cytokine expression. (A) Rats were treated ip for three days with Fyn antisense phosphothioate modified oligonucleotide (FynAS) (400 µl, 2 nmol). On the fourth day, the rats were injected via intra cava vein either with 100 µl saline (C and FynAS) or with an equal volume of leptin (10 −6 M) (Lep and FynAS+Lep); the thymuses were obtained, homogenized and 0.2 mg protein was separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-Bax or anti-Bcl-2 antibodies. (B) Isolated thymocytes were treated with leptin (10 −8 M)(Lep) or PP2 (10 −8 M) or both together and apoptosis was determined by the annexin method after 24h. (C–D) Rats were treated ip with a single dose of 100 µL of saline solution (C); 100 µL of lipopolysaccharide (LPS) 1 mg/mL; 150 µL of PP2 5 nM; 100 µL of leptin 31.2 µM (Lep) or with different combinations of these treatments; the sequence of treatment was, PP2, followed by leptin after 30 min and LPS after 30 min. Thymus was obtained after 2 h and RNA was prepared for determination of IL-1b (C) and TNF-α expression by real-time PCR. In all experiments n = 5. *p

    Article Snippet: Leptin, JAK inhibitor AG490 (tyrphostin B42) and Fyn inhibitor PP2 were from Calbiochem (La Jolla, CA, USA).

    Techniques: Inhibition, Expressing, Modification, Injection, SDS Page, Isolation, Sequencing, Real-time Polymerase Chain Reaction

    Inhibiting Fyn. (A) Rats were treated once a day for three days with a single 400 µl ip dose of buffer containing 0–8 nmol Fyn antisense (FynAS) or scrambled (FynSCR) phosphorthioate modified oligonucleotides; at the end of the experimental period the thymuses were obtained, homogenized and samples containing 0.2 mg total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-Fyn or anti-IRS1 antibodies. (B) Rats were pre-treated with a single dose of PP2 (5 nmol in 100 µl buffer, ip) (+) or saline (−), 30 min prior to leptin treatment. A single dose of leptin (100 µl 10 −6 M, via cava vein) (+), or similar volume of saline (−) was then injected; the thymuses were obtained for homogenization; samples containing 0.5 mg total protein were used in immunoprecipitation assays with anti-Fyn or anti-JAK2 antibodies; immunocomplexes were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted (IB) with anti-phosphotyrosine antibody; or 0.2 mg protein was separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-phospho ERK antibody. The depicted blots are representative of n = 5.

    Journal: PLoS ONE

    Article Title: Fyn Mediates Leptin Actions in the Thymus of Rodents

    doi: 10.1371/journal.pone.0007707

    Figure Lengend Snippet: Inhibiting Fyn. (A) Rats were treated once a day for three days with a single 400 µl ip dose of buffer containing 0–8 nmol Fyn antisense (FynAS) or scrambled (FynSCR) phosphorthioate modified oligonucleotides; at the end of the experimental period the thymuses were obtained, homogenized and samples containing 0.2 mg total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-Fyn or anti-IRS1 antibodies. (B) Rats were pre-treated with a single dose of PP2 (5 nmol in 100 µl buffer, ip) (+) or saline (−), 30 min prior to leptin treatment. A single dose of leptin (100 µl 10 −6 M, via cava vein) (+), or similar volume of saline (−) was then injected; the thymuses were obtained for homogenization; samples containing 0.5 mg total protein were used in immunoprecipitation assays with anti-Fyn or anti-JAK2 antibodies; immunocomplexes were separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted (IB) with anti-phosphotyrosine antibody; or 0.2 mg protein was separated by SDS-PAGE, transferred to nitrocellulose membranes and blotted with anti-phospho ERK antibody. The depicted blots are representative of n = 5.

    Article Snippet: Leptin, JAK inhibitor AG490 (tyrphostin B42) and Fyn inhibitor PP2 were from Calbiochem (La Jolla, CA, USA).

    Techniques: Modification, SDS Page, Injection, Homogenization, Immunoprecipitation

    VEGF induced-VE-cadherin extracellular domain cleavage is preceded by a Src-dependent VE-cadherin tyrosine phosphorylation. (A,B) HUVECs treated with VEGF (50 ng/mL) were analyzed for phosphotyrosinated-VE-cadherin in cell extracts (A) and VE-cadherin extracellular domain in conditioned medium (B): (A) VE-cadherin was immunoprecipitated from 200 µg of protein lysates and analyzed by SDS-PAGE and western blotting with the anti-phosphotyrosine antibody. VEGF induced a time-dependent tyrosine phosphorylation of VE-cadherin detectable after 2 min of stimulation. (B) Conditioned media from VEGF-stimulated HUVECs were concentrated and analyzed by SDS-PAGE and western blotting with human VE-cadherin antibody directed against VE-cadherin extracellular domain (BV9). A 90 kDa fragment corresponding to the full length VE-cadherin extracellular domain was already detectable after 10 min of VEGF stimulation. (C) HUVECs were pretreated with increasing concentrations of Src inhibitor PP2 (2.5 to 20 µM) for 15 min, prior to treatment with VEGF for 15 min. VE-cadherin was immunoprecipitated from 200 µg of protein lysates. PP2 concentrations higher than 2.5 µM completely inhibited VEGF-induced VE-cadherin tyrosine phosphorylation. (D) Analysis of conditioned media from an identical number of HUVECs pre-treated for 15 min with PP2 (5 µM) before VEGF stimulation for 15 min: the inhibitor decreased VEGF-induced VE-cadherin cleavage. (E,F) Src expression was inhibited by Src-siRNA in HUVECs 24 hours before VEGF stimulation (15min). Analysis of conditioned media showed that the knock-down of Src (controlled in F) decreased the level of soluble VE-cadherin in the media. (D,E,F) The signals were quantified using ImageJ software, error bars in graphs indicate S.D. and experiments were repeated at least three times in a similar configuration.

    Journal: PLoS ONE

    Article Title: Evidence for Post-Translational Processing of Vascular Endothelial (VE)-Cadherin in Brain Tumors: Towards a Candidate Biomarker

    doi: 10.1371/journal.pone.0080056

    Figure Lengend Snippet: VEGF induced-VE-cadherin extracellular domain cleavage is preceded by a Src-dependent VE-cadherin tyrosine phosphorylation. (A,B) HUVECs treated with VEGF (50 ng/mL) were analyzed for phosphotyrosinated-VE-cadherin in cell extracts (A) and VE-cadherin extracellular domain in conditioned medium (B): (A) VE-cadherin was immunoprecipitated from 200 µg of protein lysates and analyzed by SDS-PAGE and western blotting with the anti-phosphotyrosine antibody. VEGF induced a time-dependent tyrosine phosphorylation of VE-cadherin detectable after 2 min of stimulation. (B) Conditioned media from VEGF-stimulated HUVECs were concentrated and analyzed by SDS-PAGE and western blotting with human VE-cadherin antibody directed against VE-cadherin extracellular domain (BV9). A 90 kDa fragment corresponding to the full length VE-cadherin extracellular domain was already detectable after 10 min of VEGF stimulation. (C) HUVECs were pretreated with increasing concentrations of Src inhibitor PP2 (2.5 to 20 µM) for 15 min, prior to treatment with VEGF for 15 min. VE-cadherin was immunoprecipitated from 200 µg of protein lysates. PP2 concentrations higher than 2.5 µM completely inhibited VEGF-induced VE-cadherin tyrosine phosphorylation. (D) Analysis of conditioned media from an identical number of HUVECs pre-treated for 15 min with PP2 (5 µM) before VEGF stimulation for 15 min: the inhibitor decreased VEGF-induced VE-cadherin cleavage. (E,F) Src expression was inhibited by Src-siRNA in HUVECs 24 hours before VEGF stimulation (15min). Analysis of conditioned media showed that the knock-down of Src (controlled in F) decreased the level of soluble VE-cadherin in the media. (D,E,F) The signals were quantified using ImageJ software, error bars in graphs indicate S.D. and experiments were repeated at least three times in a similar configuration.

    Article Snippet: Reagents Leupeptin, pepstatin A, Triton X-100 were purchased from Sigma-Aldrich (Saint Louis, Missouri) and sodium orthovanadate, H2 O2 , the MMP inhibitor GM6001 from Sigma-Aldrich, and Src inhibitor PP2 from Calbiochem.

    Techniques: Immunoprecipitation, SDS Page, Western Blot, Expressing, Software

    Inhibition of Src family kinase activity blocks tyrosine phosphorylation of SLC11A1. U937-SLC11A1 cells were cultured with PMA (10ng/ml) for 48 hrs and were then left untreated (CTR) or treated with PP2 or PP3 (inactive analogue) for another 24 hours. Cell lysates were prepared. (A) c-Src and active c-Src (pY418) were monitored by Western blotting analysis. (B) Cell lysates were immunoprecipitated with the anti-c-Myc antibody 9E10, and the immunoprecipitates were probed with antibody 4G10 to phosphotyrosine for phosphorylated SLC11A1. The Western blots were stripped and re-probed with an antibody against SLC11A1. (C) The level of SLC11A1 phosphorylation was quantified by densitometry analysis and normalized to the level of total SLC11A1 protein. The relative phosphorylation level of SLC11A1 in untreated control group was set as 100%. The inhibitory effect of PP2 on SLC11A1 phosphorylation was assessed in 3 separate experiments (mean± SE). *** P

    Journal: PLoS ONE

    Article Title: c-Src kinase is involved in the tyrosine phosphorylation and activity of SLC11A1 in differentiating macrophages

    doi: 10.1371/journal.pone.0196230

    Figure Lengend Snippet: Inhibition of Src family kinase activity blocks tyrosine phosphorylation of SLC11A1. U937-SLC11A1 cells were cultured with PMA (10ng/ml) for 48 hrs and were then left untreated (CTR) or treated with PP2 or PP3 (inactive analogue) for another 24 hours. Cell lysates were prepared. (A) c-Src and active c-Src (pY418) were monitored by Western blotting analysis. (B) Cell lysates were immunoprecipitated with the anti-c-Myc antibody 9E10, and the immunoprecipitates were probed with antibody 4G10 to phosphotyrosine for phosphorylated SLC11A1. The Western blots were stripped and re-probed with an antibody against SLC11A1. (C) The level of SLC11A1 phosphorylation was quantified by densitometry analysis and normalized to the level of total SLC11A1 protein. The relative phosphorylation level of SLC11A1 in untreated control group was set as 100%. The inhibitory effect of PP2 on SLC11A1 phosphorylation was assessed in 3 separate experiments (mean± SE). *** P

    Article Snippet: SFK inhibitor PP2 and PP3 were purchased from Calbiochem (San Diego, CA).

    Techniques: Inhibition, Activity Assay, Cell Culture, Western Blot, Immunoprecipitation