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    PP2
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    Structured Review

    Millipore pp2
    PP2

    https://www.bioz.com/result/pp2/product/Millipore
    Average 99 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    pp2 - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) 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

    2) 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

    3) 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

    4) 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

    5) 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

    6) 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

    7) Product Images from "Src Inhibition Attenuates Liver Fibrosis by Preventing Hepatic Stellate Cell Activation and Decreasing Connective Tissue Growth Factor"

    Article Title: Src Inhibition Attenuates Liver Fibrosis by Preventing Hepatic Stellate Cell Activation and Decreasing Connective Tissue Growth Factor

    Journal: Cells

    doi: 10.3390/cells9030558

    Induction of autophagy is required for prevention of liver fibrosis by saracatinib. ( A ) AML12 cells were treated with saracatinib, PP2, or chloroquine for 24 h and LC3 puncta formation was analyzed by immunofluorescence. Original magnification ×800. ( B ) Representative western blot showing the effects of saracatinib (1 μM), PP2 (10 μM), and chloroquine (CQ, 10 μM) on LC3 protein levels in AML12 cells. The data in the graph are represented as the mean ± SEM of three independent measurements. * p
    Figure Legend Snippet: Induction of autophagy is required for prevention of liver fibrosis by saracatinib. ( A ) AML12 cells were treated with saracatinib, PP2, or chloroquine for 24 h and LC3 puncta formation was analyzed by immunofluorescence. Original magnification ×800. ( B ) Representative western blot showing the effects of saracatinib (1 μM), PP2 (10 μM), and chloroquine (CQ, 10 μM) on LC3 protein levels in AML12 cells. The data in the graph are represented as the mean ± SEM of three independent measurements. * p

    Techniques Used: Immunofluorescence, Western Blot

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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

    14) 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

    15) 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

    16) 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

    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 "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

    19) 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

    20) 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

    21) 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

    22) 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

    23) 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

    24) 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:

    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 "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

    27) 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

    28) 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

    29) 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

    30) 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

    31) 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

    32) 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

    33) 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

    34) 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

    35) 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

    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 "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

    38) 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

    39) 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

    40) 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

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    Negative Control:

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    Article Snippet: .. The negative control for PP2 was PP3 (4-amino-7-phenylpyrazolo[3,4-d]pyramidine) (Calbiochem) dissolved in DMSO. ..

    Mann-Whitney U-Test:

    Article Title: A Biphasic and Brain-Region Selective Down-Regulation of Cyclic Adenosine Monophosphate Concentrations Supports Object Recognition in the Rat
    Article Snippet: .. PP2 activities were expressed as nmol of phosphate released per min. Statistical differences were determined through non-parametric tests adapted to small size data (Friedman and Kruskal-Wallis, followed by a post-hoc Mann-Whitney U-test; Sigma Stat software SPSS Inc, Chicago, IL). ..

    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).

    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).

    Incubation:

    Article Title: Insulin-like growth factor binding protein-2 regulates β-catenin signaling pathway in glioma cells and contributes to poor patient prognosis
    Article Snippet: .. For treatments, cells were incubated with 250 ng/mL of Wnt3a (R & D Systems)/20 mM of LiCl/10 µM PP2 (Calbiochem)/10 µM PP3 (Calbiochem)/10 µM RGD/10 µM focal adhesion kinase (FAK) inhibitor (Sigma-Aldrich, USA)/10 µM IGF1R (Calbiochem)/500 ng/mL of IGFBP-2 (# 674-B2, R & D Systems, USA) for 24 hours. ..

    other:

    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).

    Activity Assay:

    Article Title: SRC-1 Mediates UNC-5 Signaling in Caenorhabditis elegans
    Article Snippet: .. In order to inhibit Src family kinase activity, cells were starved for 3 h in the serum-free medium and then the inhibitor PP2 (Calbiochem) was treated at the concentrations indicated in Fig. for 30 min ( ). .. Standard molecular biology techniques were used. src-1 cDNA (5′ ATGGGTTGCCTGTTTTCA…ACAAATATATATATATAA 3′) and the cytosolic domain of unc-5 cDNAs (5′ AAACGTGGCAATTCAAAA…CAAATTGTGTCCCCATAA 3′) were obtained by PCR from a worm cDNA library and were subcloned into pcDNA3myc or pcDNA3HA (Invitrogen).

    MANN-WHITNEY:

    Article Title: A Biphasic and Brain-Region Selective Down-Regulation of Cyclic Adenosine Monophosphate Concentrations Supports Object Recognition in the Rat
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    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: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Exogenous H 2 O 2 requires Src kinase to reduce surface expression of CXCR3. ( A ) The left panel shows representative FACS plots from a single donor showing the effect of H 2 O 2 on CXCR3 expression in SEB-activated T lymphocytes with or without <t>PP2</t> (1 μM) treatment. The right panel shows the mean ± SEM of three independent donors, normalized to the untreated control. Statistical significance was determined by a two-way ANOVA with a Bonferroni posttest. * p
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    Millipore signaling inhibitors pp2
    Cornea facsimiles were stained with antibody against myeloperoxidase to demonstrate granulocyte (neutrophil) infiltration after HAdV-D37 infection and leukocyte coculture. As shown ( A ), mock-infected facsimiles ( M , row 1) showed no myeloperoxidase staining, indicative of absence of infiltrating neutrophils. In virus-infected facsimiles ( V , row 2), aggregations of myeloperoxidase-positive cells ( arrows ) were observed in en face and cross sectional views, and also seen with DAPI staining ( right column ). Pretreatment with inhibitors to p38 (SB203580) and Src <t>(PP2)</t> blocked migration of leukocytes into infected facsimiles ( SB+V , row 3 , and PP2+V , row 4 ). Myeloperoxidase ( red ) signal was subsequently quantified in ImageJ by analysis of three randomly chosen fields in each of three experiments for each treatment group ( B ). Compared to DMSO, pretreatment of facsimiles with either SB203580 or PP2 prior to infection significantly reduced myeloperoxidase staining. * p
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    <t>Src</t> kinase inhibitor <t>PP2</t> blocked the incorporation of [ 3 H]-thymidine into KVL-1 cells and caused the reduction of cells in the S phase of the cell cycle. (A) KVL-1 cells (1 × 10 6 ) were treated with the Src kinase inhibitor PP2, its inactive analog
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    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

    Exogenous H 2 O 2 requires Src kinase to reduce surface expression of CXCR3. ( A ) The left panel shows representative FACS plots from a single donor showing the effect of H 2 O 2 on CXCR3 expression in SEB-activated T lymphocytes with or without PP2 (1 μM) treatment. The right panel shows the mean ± SEM of three independent donors, normalized to the untreated control. Statistical significance was determined by a two-way ANOVA with a Bonferroni posttest. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Hydrogen Peroxide Triggers a Dual Signaling Axis To Selectively Suppress Activated Human T Lymphocyte Migration

    doi: 10.4049/jimmunol.1600868

    Figure Lengend Snippet: Exogenous H 2 O 2 requires Src kinase to reduce surface expression of CXCR3. ( A ) The left panel shows representative FACS plots from a single donor showing the effect of H 2 O 2 on CXCR3 expression in SEB-activated T lymphocytes with or without PP2 (1 μM) treatment. The right panel shows the mean ± SEM of three independent donors, normalized to the untreated control. Statistical significance was determined by a two-way ANOVA with a Bonferroni posttest. * p

    Article Snippet: Chemicals PP2 is an ATP competitive inhibitor of SFKs purchased from Calbiochem.

    Techniques: Expressing, FACS

    H 2 O 2 requires an SFK to phosphorylate and enhance the catalytic activity of SHIP-1. ( A ) The left panel shows a representative FACS overlay from one donor showing the effect of H 2 O 2 on anti–phospho-SHIP-1 levels in T cells with or without PP2 treatment (1 μM). The right panel shows the mean values ± SEM from four independent donors. ( B ) Effect of H 2 O 2 (1 μM) on the catalytic activity of SHIP-1 protein immunoprecipitated from 1 × 10 6 SEB-activated T lymphocytes. Catalytic activity was quantified using a malachite green phosphatase assay, and protein concentration was determined by Western blot. Data are mean values ± SEM of three independent experiments. * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Hydrogen Peroxide Triggers a Dual Signaling Axis To Selectively Suppress Activated Human T Lymphocyte Migration

    doi: 10.4049/jimmunol.1600868

    Figure Lengend Snippet: H 2 O 2 requires an SFK to phosphorylate and enhance the catalytic activity of SHIP-1. ( A ) The left panel shows a representative FACS overlay from one donor showing the effect of H 2 O 2 on anti–phospho-SHIP-1 levels in T cells with or without PP2 treatment (1 μM). The right panel shows the mean values ± SEM from four independent donors. ( B ) Effect of H 2 O 2 (1 μM) on the catalytic activity of SHIP-1 protein immunoprecipitated from 1 × 10 6 SEB-activated T lymphocytes. Catalytic activity was quantified using a malachite green phosphatase assay, and protein concentration was determined by Western blot. Data are mean values ± SEM of three independent experiments. * p

    Article Snippet: Chemicals PP2 is an ATP competitive inhibitor of SFKs purchased from Calbiochem.

    Techniques: Activity Assay, FACS, Immunoprecipitation, Phosphatase Assay, Protein Concentration, Western Blot

    Cornea facsimiles were stained with antibody against myeloperoxidase to demonstrate granulocyte (neutrophil) infiltration after HAdV-D37 infection and leukocyte coculture. As shown ( A ), mock-infected facsimiles ( M , row 1) showed no myeloperoxidase staining, indicative of absence of infiltrating neutrophils. In virus-infected facsimiles ( V , row 2), aggregations of myeloperoxidase-positive cells ( arrows ) were observed in en face and cross sectional views, and also seen with DAPI staining ( right column ). Pretreatment with inhibitors to p38 (SB203580) and Src (PP2) blocked migration of leukocytes into infected facsimiles ( SB+V , row 3 , and PP2+V , row 4 ). Myeloperoxidase ( red ) signal was subsequently quantified in ImageJ by analysis of three randomly chosen fields in each of three experiments for each treatment group ( B ). Compared to DMSO, pretreatment of facsimiles with either SB203580 or PP2 prior to infection significantly reduced myeloperoxidase staining. * p

    Journal: In vitro cellular & developmental biology. Animal

    Article Title: Novel model of innate immunity in corneal infection

    doi: 10.1007/s11626-015-9910-2

    Figure Lengend Snippet: Cornea facsimiles were stained with antibody against myeloperoxidase to demonstrate granulocyte (neutrophil) infiltration after HAdV-D37 infection and leukocyte coculture. As shown ( A ), mock-infected facsimiles ( M , row 1) showed no myeloperoxidase staining, indicative of absence of infiltrating neutrophils. In virus-infected facsimiles ( V , row 2), aggregations of myeloperoxidase-positive cells ( arrows ) were observed in en face and cross sectional views, and also seen with DAPI staining ( right column ). Pretreatment with inhibitors to p38 (SB203580) and Src (PP2) blocked migration of leukocytes into infected facsimiles ( SB+V , row 3 , and PP2+V , row 4 ). Myeloperoxidase ( red ) signal was subsequently quantified in ImageJ by analysis of three randomly chosen fields in each of three experiments for each treatment group ( B ). Compared to DMSO, pretreatment of facsimiles with either SB203580 or PP2 prior to infection significantly reduced myeloperoxidase staining. * p

    Article Snippet: In select experiments, cells were treated overnight with the signaling inhibitors PP2 or SB203580 (10 μM each) (Calbiochem, La Jolla, CA) prior to incubation with human peripheral blood leukocytes.

    Techniques: Staining, Infection, Migration

    Src kinase inhibitor PP2 blocked the incorporation of [ 3 H]-thymidine into KVL-1 cells and caused the reduction of cells in the S phase of the cell cycle. (A) KVL-1 cells (1 × 10 6 ) were treated with the Src kinase inhibitor PP2, its inactive analog

    Journal: Blood

    Article Title: Activation of Src kinase Lyn by the Kaposi sarcoma-associated herpesvirus K1 protein: implications for lymphomagenesis

    doi: 10.1182/blood-2004-07-2781

    Figure Lengend Snippet: Src kinase inhibitor PP2 blocked the incorporation of [ 3 H]-thymidine into KVL-1 cells and caused the reduction of cells in the S phase of the cell cycle. (A) KVL-1 cells (1 × 10 6 ) were treated with the Src kinase inhibitor PP2, its inactive analog

    Article Snippet: When required, the Src kinase inhibitor PP2, the Syk kinase inhibitor piceatannol, and the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 (Calbiochem, La Jolla, CA) were added to the cell culture medium at the indicated concentrations 4 hours before harvest.

    Techniques:

    Inhibition of constitutive NF-κB promoter activity and VEGF production by Src kinase inhibitor PP2 in K1 lymphoma–derived KVL-1 cells. KVL-1 cells (2 × 10 6 ) that express K1 were transfected with 10 μg of NF-κB–luciferase

    Journal: Blood

    Article Title: Activation of Src kinase Lyn by the Kaposi sarcoma-associated herpesvirus K1 protein: implications for lymphomagenesis

    doi: 10.1182/blood-2004-07-2781

    Figure Lengend Snippet: Inhibition of constitutive NF-κB promoter activity and VEGF production by Src kinase inhibitor PP2 in K1 lymphoma–derived KVL-1 cells. KVL-1 cells (2 × 10 6 ) that express K1 were transfected with 10 μg of NF-κB–luciferase

    Article Snippet: When required, the Src kinase inhibitor PP2, the Syk kinase inhibitor piceatannol, and the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 (Calbiochem, La Jolla, CA) were added to the cell culture medium at the indicated concentrations 4 hours before harvest.

    Techniques: Inhibition, Activity Assay, Derivative Assay, Transfection, Luciferase