Journal: Journal of Neuroinflammation

Article Title: Hyperammonemia alters membrane expression of GluA1 and GluA2 subunits of AMPA receptors in hippocampus by enhancing activation of the IL-1 receptor: underlying mechanisms

doi: 10.1186/s12974-018-1082-z

Figure Lengend Snippet: ” section. Values are expressed as percentage of basal levels in control rats and are the mean ± SEM of 15 rats per group in a , b , and c . Data were analyzed by two-way ANOVA. In a , F (1, 55) = 1.077 for effect of HA, p = 0.3039; F (1, 55) = 1.220 for effect of IL-1Ra, p = 0.2742; and F (1, 55) = 14.93 for interaction, p = 0.0003. In b , F (1, 56) = 2.647 for effect of HA, p = 0.1093; F (1, 56) = 15.57 for effect of PP2, p = 0.0002; and F (1, 56) = 1.630 for interaction, p = 0.2069. In c , F (1, 55) = 10.21 for effect of HA, p = 0.0023; F (1, 55) = 30.59 for effect of ifenprodil, p

Article Snippet: One hundred nanogram per millileter of IL1Ra (R & D Systems cat# 1545-RA-025, Minneapolis, USA), 100 μM ifenprodil (Abcam cat# ab120111, Cambridge, MA), 20 μM SB239063 (Tocris cat# 1962, Bristol, UK), 10 μM rottlerin (Sigma cat# R5648, Darmstadt, Germany), or 10 μM PP2 (Sigma cat# P0042, Darmstadt, Germany) was added to the incubation buffer during 30 min to specifically inhibit IL-1 receptor, GluN2B, p38, PKCδ, and Src activities, respectively.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Hyperammonemia alters membrane expression of GluA1 and GluA2 subunits of AMPA receptors in hippocampus by enhancing activation of the IL-1 receptor: underlying mechanisms

doi: 10.1186/s12974-018-1082-z

Figure Lengend Snippet: Enhanced activation of IL-1 receptor and Src leads to increased phosphorylation of GluN2B subunit at Ser1303 and reduced membrane-associated CaMKII in hyperammonemic rats. IL-1Ra or PP2 were added to hippocampal slices. CaMKII membrane association ( a , b ) and phosphorylation of GluN2B subunit at Ser1303 ( c , d ” section. Values are expressed as percentage of basal levels in control rats and are the mean ± SEM of 12, 15, 17, and 17 rats per group in a , b , c , and d respectively. Data were analyzed by two-way ANOVA. In a , F (1, 45) = 0.2872 for effect of HA, p = 0.5947; F (1, 45) = 1.742 for effect of IL-1Ra, p = 0.1936; and F (1, 45) = 7.909 for interaction, p = 0.0073. In b , F (1, 54) = 0.4278 for effect of HA, p = 0.5159; F (1, 54) = 18.16 for effect of PP2, p

Article Snippet: One hundred nanogram per millileter of IL1Ra (R & D Systems cat# 1545-RA-025, Minneapolis, USA), 100 μM ifenprodil (Abcam cat# ab120111, Cambridge, MA), 20 μM SB239063 (Tocris cat# 1962, Bristol, UK), 10 μM rottlerin (Sigma cat# R5648, Darmstadt, Germany), or 10 μM PP2 (Sigma cat# P0042, Darmstadt, Germany) was added to the incubation buffer during 30 min to specifically inhibit IL-1 receptor, GluN2B, p38, PKCδ, and Src activities, respectively.

Techniques: Activation Assay

Journal: Journal of Neuroinflammation

Article Title: Hyperammonemia alters membrane expression of GluA1 and GluA2 subunits of AMPA receptors in hippocampus by enhancing activation of the IL-1 receptor: underlying mechanisms

doi: 10.1186/s12974-018-1082-z

Figure Lengend Snippet: IL-1 receptor-mediated activation of Src leads to the alterations in membrane expression and phosphorylation of GluA1 and GluA2 subunit in hyperammonemic rats. IL-1Ra or PP2, an inhibitor of Src kinase, were added to hippocampal slices. Phosphorylation of Src at Tyr416 ( a ), of GluA1 at Ser831 ( c ), of GluA2 at Ser880 ( e ), and membrane expression of GluA1 ( b ) and GluA2 ( d section. Values are expressed as percentage of basal levels in control rats and are the mean ± SEM of 24, 22, 33, 32, and 18 rats per group in a , b , c , d , and e respectively. Data were analyzed by two-way ANOVA. In a , F (1, 92) = 0.005730 for effect of HA, p = 0.9398; F (1, 92) = 5.434 for effect of IL1Ra, p = 0.0219; and F (1, 92) = 3.058 for interaction, p = 0.0837. In b , F (1, 83) = 1.361 for effect of HA, p = 0.2466; F (1, 83) = 9.148 for effect of PP2, p = 0.0033; and F (1, 83) = 8.418 for interaction, p = 0.0048. In c , F (1, 127) = 4.230 for effect of HA, p = 0.0418; F (1, 127) = 5.912 for effect of PP2, p = 0.0164; and F (1, 127) = 2.146 for interaction, p = 0.1454. In d , F (1, 123) = 1.545 for effect of HA, p = 0.2162; F (1, 123) = 8.038 for effect of PP2, p = 0.0054; and F (1, 123) = 11.17 for interaction, p = 0.0011. In e , F (1, 68) = 7.756 for effect of HA, p = 0.0069; F (1, 68) = 7.864 for effect of PP2, p = 0.0066; and F (1, 68) = 44.50 for interaction, p

Article Snippet: One hundred nanogram per millileter of IL1Ra (R & D Systems cat# 1545-RA-025, Minneapolis, USA), 100 μM ifenprodil (Abcam cat# ab120111, Cambridge, MA), 20 μM SB239063 (Tocris cat# 1962, Bristol, UK), 10 μM rottlerin (Sigma cat# R5648, Darmstadt, Germany), or 10 μM PP2 (Sigma cat# P0042, Darmstadt, Germany) was added to the incubation buffer during 30 min to specifically inhibit IL-1 receptor, GluN2B, p38, PKCδ, and Src activities, respectively.

Techniques: Activation Assay, Expressing

Journal: Journal of Neuroinflammation

Article Title: Hyperammonemia alters membrane expression of GluA1 and GluA2 subunits of AMPA receptors in hippocampus by enhancing activation of the IL-1 receptor: underlying mechanisms

doi: 10.1186/s12974-018-1082-z

Figure Lengend Snippet: IL-1 receptor, Src, and GluN2B-mediated activation of p38 leads to the alterations in membrane expression and phosphorylation of the GluA2 but not of the GluA1 subunit in hyperammonemic rats. IL-1Ra, PP2, SB239063, an inhibitor of p38 MAP-kinase, or ifenprodil, an antagonist of GluN2B-containing NMDA receptors, were added to hippocampal slices. Phosphorylation of p38 ( a , b , c ), GluA1 at Ser831 ( g ), and GluA2 at Ser880 ( e ) and membrane expression of GluA1 ( f ) and GluA2 ( d section. Values are expressed as percentage of basal levels in control rats and are the mean ± SEM of 18, 16, 20, 30, 17, 22, and 29 rats per group in a , b , c , d , e , f , and g respectively. Data were analyzed by two-way ANOVA. In a , F (1, 66) = 0.004793 for effect of HA, p = 0.9450; F (1, 66) = 23.42 for effect of IL-1Ra, p

Article Snippet: One hundred nanogram per millileter of IL1Ra (R & D Systems cat# 1545-RA-025, Minneapolis, USA), 100 μM ifenprodil (Abcam cat# ab120111, Cambridge, MA), 20 μM SB239063 (Tocris cat# 1962, Bristol, UK), 10 μM rottlerin (Sigma cat# R5648, Darmstadt, Germany), or 10 μM PP2 (Sigma cat# P0042, Darmstadt, Germany) was added to the incubation buffer during 30 min to specifically inhibit IL-1 receptor, GluN2B, p38, PKCδ, and Src activities, respectively.

Techniques: Activation Assay, Expressing

Journal: Journal of Neuroinflammation

Article Title: Hyperammonemia alters membrane expression of GluA1 and GluA2 subunits of AMPA receptors in hippocampus by enhancing activation of the IL-1 receptor: underlying mechanisms

doi: 10.1186/s12974-018-1082-z

Figure Lengend Snippet: IL-1 receptor and Src activation lead to increased phosphorylation at Tyr1472 and membrane expression of the GluN2B subunit in hyperammonemic rats. IL-1Ra or PP2, an inhibitor of Src kinase, were added to hippocampal slices. Phosphorylation of GluN2B at Tyr1472 ( a , c ) and membrane expression of GluN2B ( b , d section. Values are expressed as percentage of basal levels in control rats and are the mean ± SEM of 27, 37, 28, and 37 rats per group in a , b , c , and d respectively. Data were analyzed by two-way ANOVA. In a , F (1, 104) = 0.3170 for effect of HA, p = 0.5746; F (1, 104) = 5.592 for effect of IL-1Ra, p = 0.0199; and F (1, 104) = 9.208 for interaction, p = 0.0030. In b , F (1, 143) = 3.642 for effect of HA, p = 0.0583; F (1, 143) = 10.33 for effect of IL-1Ra, p = 0.0016; and F (1, 143) = 9.704 for interaction, p = 0.0022. In c , F (1, 106) = 1.727 for effect of HA, p = 0.1917; F (1, 106) = 6.991 for effect of PP2, p = 0.0094; and F (1, 106) = 1.457 for interaction, p = 0.2302. In d , F (1, 143) = 8.814 for effect of HA, p = 0.0035; F (1, 143) = 5.432 for effect of PP2, p = 0.0212; and F (1, 143) = 4.963 for interaction, p = 0.0275. Values significantly different from control rats are indicated by asterisk and from hyperammonemic rats are indicated by “a”. Bonferroni post-test: * p

Article Snippet: One hundred nanogram per millileter of IL1Ra (R & D Systems cat# 1545-RA-025, Minneapolis, USA), 100 μM ifenprodil (Abcam cat# ab120111, Cambridge, MA), 20 μM SB239063 (Tocris cat# 1962, Bristol, UK), 10 μM rottlerin (Sigma cat# R5648, Darmstadt, Germany), or 10 μM PP2 (Sigma cat# P0042, Darmstadt, Germany) was added to the incubation buffer during 30 min to specifically inhibit IL-1 receptor, GluN2B, p38, PKCδ, and Src activities, respectively.

Techniques: Activation Assay, Expressing

Journal: Scientific Reports

Article Title: Neutrophil elastase promotes macrophage cell adhesion and cytokine production through the integrin-Src kinases pathway

doi: 10.1038/s41598-020-72667-3

Figure Lengend Snippet: NE activates cytokine production through Src kinases pathway. ( A ) Time dependent phosphorylation of Src kinases in response to NE treatment. ( B ) Pretreatment of Mɸ with PP2 abolished Src phosphorylation by NE treatment. Representative images of 3 independent experiments. ( C ) The treatment with PP2 (2 µM) blocked NE-induced IL-8, IL-1β, and TNFα cytokine production. * p

Article Snippet: Collagen Type I from rat tail and Src inhibitor PP2 were obtained from EMD Millipore (Burlington, MA, USA).

Techniques:

Journal: EMBO Molecular Medicine

Article Title: The MICA-129 dimorphism affects NKG2D signaling and outcome of hematopoietic stem cell transplantation

doi: 10.15252/emmm.201505246

Figure Lengend Snippet: NKG2D binding to the MICA-129Met and MICA-129Val isoform and triggering of phosphorylation of SRC family kinases The linear regression of MICA expression intensity and binding of a recombinant NKG2D-Fc fusion protein both determined as MFI by flow cytometry is displayed for L-MICA-129Met ( n = 79, left panel) and L-MICA-129Val clones ( n = 81, right panel). The coefficients of determination ( R 2 ), the regression coefficients (reg. coeff.), and the P -values for Pearson correlation are indicated. Purified IL-2-stimulated (100 U/ml for 4 days) NK cells (10 6 ) were stimulated with immobilized MICA-129Met-mIgG 2a -Fc or MICA-129Val-mIgG 2a -Fc or OVA-mIgG 2a -Fc fusion proteins (10 μg/ml) for 3, 10, or 30 min. The protein lysates of these cells were separated by SDS–PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb, an anti-phospho-SRC family (Tyr419) kinases Ab, and an anti-β-actin mAb as a loading control. The arrow points toward phosphorylated SRC family kinases. Blots obtained from three independent experiments were analyzed by densitometry, and the means plus SD of the ratio between phospho-SRC family kinase and β-actin signals is displayed. The difference between NK cells stimulated for 10 min by MICA-129Met-Fc or MICA-129Val-Fc proteins was assessed by t -test. Purified IL-2-stimulated NK cells (100 U/ml for 4 days, 10 6 ) were incubated with the SRC kinase inhibitor PP2 (25 μM), the vehicle DMSO, or medium only (Ø) for 30 min before being added to immobilized MICA-129Met-Fc, MICA-129Val-Fc, or OVA-Fc fusion proteins (10 μg/ml) for 10 min. The protein lysates of these cells were separated by SDS–PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb and an anti-β-actin mAb as a loading control. The blot is representative for two independent experiments. In parallel, degranulation of the NK cells was measured by anti-CD107a staining in flow cytometry. The difference between DMSO- and PP2-treated cells with respect to CD107a + cells and the MFI of CD107a is indicated in the histograms. The results are representative for two independent experiments.

Article Snippet: A potential cytotoxic effect of the SRC kinase inhibitor PP2 (25 μM; Sigma-Aldrich, #P0042) was determined by staining of NK cells with Annexin V-FITC (BD Biosciences) in combination with PI in binding buffer according to the manufacturer’s protocol.

Techniques: Binding Assay, Expressing, Recombinant, Flow Cytometry, Cytometry, Clone Assay, Purification, SDS Page, Incubation, Staining

Journal: Nature neuroscience

Article Title: Activation of FAK and Src are receptor-proximal events required for netrin signaling

doi: 10.1038/nn1329

Figure Lengend Snippet: Src activity is required for netrin-induced axon growth. ( a ) PP2 inhibits netrin-stimulated axon growth. Explants from the dorsal half of E12 spinal cords were cultured in collagen gels for 20 h. Netrin-1-induced outgrowth of axon bundles was inhibited by PP2 but not PP3. Scale bar, 100 μm. ( b ) PP2 inhibits netrin-stimulated axon growth. The netrin (20 h) and PP2 (0.2 μM) treatments are indicated (netrin versus control, P

Article Snippet: To inhibit SFK activity, the inhibitor PP2 (Calbiochem) was added to serum-starved cells at a concentration of 0.2–5 μM in 1% DMSO for 1 h. Tyrosine phosphorylation was determined by immunoblotting using PY20 antibody.

Techniques: Activity Assay, Cell Culture

Journal: Nature neuroscience

Article Title: Activation of FAK and Src are receptor-proximal events required for netrin signaling

doi: 10.1038/nn1329

Figure Lengend Snippet: Src interacts with DCC and is activated by netrin. ( a ) Src activates DCC tyrosine phosphorylation. HEK293 cells were transfected with DCC (500 ng) and Src (50 ng) and treated with PP2 as indicated. Tyrosine phosphorylation of DCC was determined by anti-DCC IP followed by anti-PY20 IB. ( b ) Netrin stimulates Src. Cells were transfected with DCC (250 ng) and Src (10 ng) and stimulated with netrin. Tyrosine phosphorylation of DCC and Src were determined using anti-pY20 and Src-Y418, respectively. ( c ) Synergistic tyrosine phosphorylation of DCC by Src and FAK. DCC was cotransfected with various amounts of FAK and Src as indicated. Tyrosine phosphorylation of DCC was determined. ( d ) Co-IP of Src with DCC. HEK293 cells were transfected as indicated and subjected to IP with either anti-DCC (left) or anti-Src (right). Co-IP of Src (left) or DCC (right) was determined by IB. ( e ) Interaction of endogenous Src and DCC. Endogenous Src was immunoprecipitated. The presence of endogenous DCC in the Src IP was determined by IB. IP with anti-GAL-4 and anti-Src incubated with buffer only were used as negative controls. ( f ) Netrin enhances the interaction between DCC and Src in transfected HEK293 cells. Src was immunoprecipitated by Src antibody and the IP was probed with DCC antibody for DCC. ( g ) Netrin stimulates Src tyrosine phosphorylation and kinase activity in the E12 mouse brain cortex neurons. Left, Src tyrosine phosphorylation; right, quantification of kinase activity after 10 min and 20 min of netrin application. ( h ) Netrin stimulates Fyn tyrosine phosphorylation and kinase activity. Experiments are similar to those in g . ( i ) Netrin did not induce DCC tyrosine phosphorylation in Src/Fyn/Yes (S/F/Y) triple-knockout MEF cells. DCC and UNC-5 were cotransfected into wild-type (WT) and Src/Fyn/Yes triple knockout MEFs, and then netrin-induced DCC tyrosine phosphorylation was determined.

Article Snippet: To inhibit SFK activity, the inhibitor PP2 (Calbiochem) was added to serum-starved cells at a concentration of 0.2–5 μM in 1% DMSO for 1 h. Tyrosine phosphorylation was determined by immunoblotting using PY20 antibody.

Techniques: Droplet Countercurrent Chromatography, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, Incubation, Activity Assay, Triple Knockout

Journal: Nature neuroscience

Article Title: Activation of FAK and Src are receptor-proximal events required for netrin signaling

doi: 10.1038/nn1329

Figure Lengend Snippet: Src function or DCC tyrosine phosphorylation is required for DCC-mediated axon attraction. ( a ) Superimposed neurite trajectories of DCC-expressing neurons in the presence of 0.1 μM PP2 or PP3 during 1-h exposure to a netrin gradient (2.5 μg/ml in the pipette, arrows) for all the neurons examined. Arrows, presence of netrin gradient or buffer control. ‘PP2’ and ‘PP3,’ presence of 0.1 μM PP2 and PP3, respectively, in the presence of a netrin gradient. ( b ) Cumulative distribution of turning angles. Significant differences from the control groups are marked (* P

Article Snippet: To inhibit SFK activity, the inhibitor PP2 (Calbiochem) was added to serum-starved cells at a concentration of 0.2–5 μM in 1% DMSO for 1 h. Tyrosine phosphorylation was determined by immunoblotting using PY20 antibody.

Techniques: Droplet Countercurrent Chromatography, Expressing, Transferring

Journal: Cardiology Research and Practice

Article Title: Influence of Endogenous Cardiac Glycosides, Digoxin, and Marinobufagenin in the Physiology of Epithelial Cells

doi: 10.1155/2019/8646787

Figure Lengend Snippet: Participation of c-Src and ERK1/2 in the signaling pathway that leads to GJIC and TER increase by treatment with digoxin and marinobufagenin. (a, b) Pretreatment of monolayers of MDCK-W with inhibitors of c-Src (PP2, 10 μ M) and ERK1/2 (PD98059, 25 μ M) suppresses the enhancement of GJIC (a(I, II) and b(I, II)) and TER (a(III) and b(III)) induced by digoxin (a) or marinobufagenin (b) in MDCK cells in mature monolayers. (I, II) Effect of inhibitors on GJIC. (I) Series of representative images of dye transfer trial of each treatment assayed. (II) A bar chart comparing the average number of cells stained on each condition, as indicated below by each bar. The number of repeats is indicated in the upper part of each bar. Simple, paired comparisons (Student's t ) were made between treatment conditions, as indicated by the lines above bars. ∗ p ≤ 0.05; ∗∗ p ≤ 0.001. (III) Bar charts comparing the mean ± SE ( n = 9). Value of TER (Ohms·cm 2 ) at different treatment conditions, as indicated in the inset, at 24, 48, and 72 hours of treatment ∗ p ≤ 0.05; ∗∗ p ≤ 0.005 of simple, pairwise comparisons (Student's t ) of TER values obtained from monolayers treated with digoxin or marinobufagenin in the presence or absence of PD or PP2.

Article Snippet: Lucifer Yellow was obtained from Sigma-Aldrich (67764-47-5) In the corresponding assays, cells were exposed to 10 μ M PP2, an inhibitor of c-Src kinase (MEK-1; 513000; Merk Millipore, Darmstadt GE) and 25 μ M PD98059, an inhibitor of mitogen extracellular kinase-1 (529573; Merk Millipore).

Techniques: Staining

Journal: Oncotarget

Article Title: CD93 and dystroglycan cooperation in human endothelial cell adhesion and migration

doi: 10.18632/oncotarget.7136

Figure Lengend Snippet: Binding of DG to laminin induces CD93 phosphorylation via Src ( A ) The schematic diagram illustrates the domains of CD93 and the 47-amino acid sequence of its cytoplasmic tail containing tyrosine 628 and 644. CTLD, C-type lectin-like domain; EGF-like, Epidermal Growth Factor repeats; Mucin. Mucin-like domain; TM, transmembrane domain. ( B ) Cell extracts from ECs spreading and growing on gelatin or laminin were immunoprecipitated with anti-CD93 antibodies. Immunoprecipitates were analyzed by Western blotting with anti-phosphotyrosine and anti-CD93 antibodies to confirm equal loading. ( C ) HUVEC were infected with a lentiviral vector expressing unrelated (unr) or DG (clones C7 or C10) shRNAs. Not infected ECs were also analyzed (HUVEC). Cell extracts from cells spreading and growing on laminin were immunoprecipitated or not (+, −) with anti-CD93 antibodies and analyzed by Western blotting with anti-phosphotyrosine antibodies. To confirm equal loading, whole cell lysates were analyzed by Western blotting with anti-CD93 and anti-β-actin antibodies. ( D ) HUVEC were allowed to adhere and grow on laminin in the presence (+) or not (−) of PP2 (10 μm). Cell lysates were immunoprecipitated with anti-CD93 antibodies and analyzed by immunoblotting with anti-phosphotyrosine and anti-CD93 antibodies to confirm equal loading. In the same cell lysates, phosphorylation on tyrosine 416 of Src, a protein modification that is closely correlated with kinase activity, was analyzed by Western blotting with anti-phospho-Y416 Src and anti-Src antibodies to confirm equal loading. All experiments were performed three-four times. ( E ) Human Lenti-X 293T cells, which do not express wild type CD93, were transiently cotransfected with a construct expressing human CD93 and the constitutively active (DP) or kinase dead (DN) Src kinase. Transfection with an empty vector (mock) is indicated. Cell lysates were immunoprecipitated with anti-CD93 antibodies and analyzed by Western blotting with anti-phosphotyrosine and anti-CD93 antibodies to confirm equal loading.

Article Snippet: The Src family tyrosine kinase inhibitor PP2 was purchased from Calbiochem (San Diego, CA, USA).

Techniques: Binding Assay, Sequencing, Immunoprecipitation, Western Blot, Infection, Plasmid Preparation, Expressing, Modification, Activity Assay, Construct, Transfection

Journal: Oncogenesis

Article Title: Targeting Src in endometriosis-associated ovarian cancer

doi: 10.1038/oncsis.2016.54

Figure Lengend Snippet: In vitro inhibition of Src signaling in anchorage-independent and anchorage-dependent growth assays. ( a–e ) Relative number of colonies in ( a ) Hey.A8, ( b , c ) A2780.cp, ( d ) HEY-C2, ( e ) HAC-2 and TOV21G cell lines. ( c ) Representative A2780.cp colonies in cultures treated with vehicle or 50 μ m PP2 are shown. Data shown are mean±s.d. of three independent experiments. ( g ) Three Src-pY416-positive and one Src-pY416-negative cell line were assayed for anchorage-dependent growth when treated with PP2. Cells were cultured in the presence of PP2 for 7 days.

Article Snippet: Anchorage-independent growth assays Assays were performed with PP2 (Tocris Bioscience, Pittsburgh, PA, USA) at doses of 50 μm, 5 μm, 500 nm and 5 nm.

Techniques: In Vitro, Inhibition, HAC Assay, Cell Culture

Journal: Oncogenesis

Article Title: Targeting Src in endometriosis-associated ovarian cancer

doi: 10.1038/oncsis.2016.54

Figure Lengend Snippet: Inhibition of Src signaling in 3D OC models. Three Src-pY416-positive and one Src-pY416-negative cell lines were cultured as 3D models in the presence of various doses of PP2. Cultured were fixed after 7 days of exposure. Phase images and H E sections enable qualitative assessment of the effects of PP2.

Article Snippet: Anchorage-independent growth assays Assays were performed with PP2 (Tocris Bioscience, Pittsburgh, PA, USA) at doses of 50 μm, 5 μm, 500 nm and 5 nm.

Techniques: Inhibition, Cell Culture

Journal: Journal of Inflammation (London, England)

Article Title: Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages

doi: 10.1186/1476-9255-3-8

Figure Lengend Snippet: Zymosan and bacteria but not LPS or peptidoglycan (PGN) induce phosphorylation of Btk . ( A ) Macrophages were stimulated with zymosan (zym), S.aureus ( S.a .), LPS or PGN for 30 min. (B and C) Macrophages were pretreated for 15 min with either wortmannin (W, 100 nM), PP2 (5 μM), SU6656 (5 μM) or SKI-1 (5 μM), followed by stimulation with zymosan ( B ) or S.aureus ( C ) for 30 min. Equal amounts of cell lysate were run on polyacrylamide gels and probed with phosphospecific antibodies against Btk. The membrane was reprobed with ERK-2 antibody to verify equal loading of proteins. The data are representative of three separate experiments.

Article Snippet: PP2 was from Biomol (Plymouth Meeting, PA, USA), while SP600125 was from Tocris Cockson (Northpoint, UK).

Techniques:

Journal: Journal of Inflammation (London, England)

Article Title: Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages

doi: 10.1186/1476-9255-3-8

Figure Lengend Snippet: Inhibition by PP2 or SKI-1 of zymosan- and S.aureus - induced phosphorylation of MAP kinases . Macrophages were pretreated for 15 min with PP2 (1–10 μM), followed by stimulation with zymosan ( A ) or S.aureus ( B ) for 20 min. ( C ) Macrophages were pretreated for 15 min with SKI-1 (5 μM), followed by stimulation with zymosan or S.aureus for 20 min. Equal amounts of cell lysate were run on 10% polyacrylamide gels and probed with phosphospecific antibodies against ERK, p38 and JNK. The membrane was reprobed with ERK-2 antibody to verify equal loading of protein.

Article Snippet: PP2 was from Biomol (Plymouth Meeting, PA, USA), while SP600125 was from Tocris Cockson (Northpoint, UK).

Techniques: Inhibition

Journal: Journal of Inflammation (London, England)

Article Title: Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages

doi: 10.1186/1476-9255-3-8

Figure Lengend Snippet: Effect of inhibitors against PI3K, Src family kinases and Btk on the tyrosine phosphorylation of Akt . Zymosan but not S. aureus induce tyrosine phosphorylation of Akt. This phosphorylation was affected by inhibitors against PI3K and Src family kinases. (A and C) Macrophages were stimulated for 30 min with either zymosan or heat killed S.aureus . (B) Macrophages were pretreated for 15 min with either PP2 (5 μM) or wortmannin (W, 100 nM), followed by stimulation with zymosan for 30 min. Cell lysates were processed for immunoblotting with the indicated antibody as described in Methods. The membrane was reprobed with ERK-2 antibody to verify equal loading of proteins. The data are representative of three separate experiments.

Article Snippet: PP2 was from Biomol (Plymouth Meeting, PA, USA), while SP600125 was from Tocris Cockson (Northpoint, UK).

Techniques:

Journal: Journal of Inflammation (London, England)

Article Title: Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages

doi: 10.1186/1476-9255-3-8

Figure Lengend Snippet: Zymosan but not S.aureus induced tyrosine phosphorylation of PLC γ2 . ( A ) Macrophages were stimulated with zymosan(z) or S.aureus ( S.a ) for 45 min. (B and C) Macrophages were pretreated for 15 min with either PP2 (5 μM), wortmannin (W, 100 nM) ( B ) or SKI-1 (5 μM) ( C ) followed by stimulation with zymosan for 30 min. Cell lysates were immunoprecipitated with antibody against PLCγ2 as described, followed by Western blot analysis with phosphotyrosine-specific antibody. The membrane was stripped and reprobed with antibody against PLCγ2. The data are representative of three separate experiments.

Article Snippet: PP2 was from Biomol (Plymouth Meeting, PA, USA), while SP600125 was from Tocris Cockson (Northpoint, UK).

Techniques: Planar Chromatography, Immunoprecipitation, Western Blot

Journal: Journal of Inflammation (London, England)

Article Title: Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages

doi: 10.1186/1476-9255-3-8

Figure Lengend Snippet: Inhibition by PP2 or SKI-1 of zymosan- and S.aureus -induced release of arachidonate . Macrophages were labeled with [3H]arachidonic acid for 20 h. The cells were pretreated for 15 min with the indicated concentrations of PP2 (A) or SKI-1 (B) followed by stimulation with either zymosan (●) for 45 min or S.aureus (▲) for 60 min. Results are mean ± SEM (n = 3) and corrected for the release in control cultures.

Article Snippet: PP2 was from Biomol (Plymouth Meeting, PA, USA), while SP600125 was from Tocris Cockson (Northpoint, UK).

Techniques: Inhibition, Labeling

Journal: Frontiers in Immunology

Article Title: High-Affinity Ligands Can Trigger T Cell Receptor Signaling Without CD45 Segregation

doi: 10.3389/fimmu.2018.00713

Figure Lengend Snippet: Calcium flux in Jurkat T cells stimulated with membrane-tethered anti-CD3 scFv. Jurkat T cells were dropped on planar lipid bilayers decorated with anti-CD3 scFv in the presence or absence of Lck inhibitor (PP2). The emission was measured from Fura-2AM labeled Jurkat T cells as the ratio between 340 and 380 nm at different times (min). (A) Ca 2+ flux measured from individual Fura-2AM stained Jurkat T cells. A heat map for Ca 2+ from individual Jurkat T cells over time. Mean ratiometric fluorescence of Fura-2AM loaded Jurkat T cells triggered by membrane-tethered OKT3-1 (B) , OKT3-CD43 (C) , OKT3 MA -1 (D) , OKT3 MA -CD43 (E) , or anti-DNS control scFv (F) ( n = 52 and 30 cells without PP2 and with PP2 inhibition, respectively). Bars, SD.

Article Snippet: The specificity of the calcium response was ensured by pretreating Jurkat T cells with 25 µM PP2 (Abcam, Cambridge, MA, USA), a potent inhibitor of p56lck tyrosine phosphorylation, for 30 min at 37°C before dropping the cells.

Techniques: Labeling, Staining, Fluorescence, Inhibition

Journal: Molecular and cellular endocrinology

Article Title: Phosphorylation of Ser-279/282 and Tyr-265 Positions on Cx43 as Possible Mediators of VEGF-165 Inhibition of Pregnancy-Adapted Ca2+ Burst Function in Ovine Uterine Artery Endothelial Cells

doi: 10.1016/j.mce.2015.05.030

Figure Lengend Snippet: Effect of PMA, ATP and VEGF-165 and the inhibition by PP2 on Cx43 phosphorylation

Article Snippet: PP2 (a selective inhibitor of Src family kinases) was from Enzo Life Sciences.

Techniques: Inhibition

Journal: Molecular and cellular endocrinology

Article Title: Phosphorylation of Ser-279/282 and Tyr-265 Positions on Cx43 as Possible Mediators of VEGF-165 Inhibition of Pregnancy-Adapted Ca2+ Burst Function in Ovine Uterine Artery Endothelial Cells

doi: 10.1016/j.mce.2015.05.030

Figure Lengend Snippet: Effect of PMA, ATP and VEGF-165 and the inhibition by PP2 on ERK phosphorylation

Article Snippet: PP2 (a selective inhibitor of Src family kinases) was from Enzo Life Sciences.

Techniques: Inhibition

Journal: Cell Death & Disease

Article Title: Novel β-phenylacrylic acid derivatives exert anti-cancer activity by inducing Src-mediated apoptosis in wild-type KRAS colon cancer

doi: 10.1038/s41419-018-0942-x

Figure Lengend Snippet: Activation of Src mediates MHY-induced apoptosis. a Caco2 and HT29 cells were treated with MHYs (10 μM) for 24 h and xenografts (HT29 cells) were treated with MHYs (3 mg/kg) for 8 days. Src phosphorylation levels were measured in Caco2 and HT29 cells as well as in xenografts. b Caco2 and HT29 cells were treated with MHYs (10 μM) for 24 h to investigate the levels of phospho-JNK and phospho-c-Jun. c HT29 cells were transfected with scrambled siRNA and Src siRNA. d HT29 cells expressing scrambled siRNA and Src siRNA were treated with MHYs (10 μM) for 24 h to measure JNK phosphorylation. e HT29 cells were pre-treated with the Src inhibitor PP2 (100 nM) for 30 min followed by MHYs (10 μM) for 24 h. Relative density measurements correspond to the intensities of the immunoblotting bands normalized to an internal control ( n = 3). f Mitochondrial membrane dysfunction was determined by FACS analysis. HT29 cells were pre-treated with PP2 (100 nM) for 30 min followed by MHYs (10 μM) for 24 h ( n = 3). g HT29 cells expressing scrambled siRNA and Src siRNA were treated with MHYs (10 μM) for 24 h to measure mitochondrial dysfunction ( n = 3). Data are shown as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey post-hoc test. Statistical significance is indicated as ** p

Article Snippet: PP2 (Cayman Chemical), Src Tyr kinase inhibitor, was dissolved in DMSO.

Techniques: Activation Assay, Transfection, Expressing, FACS

Journal: PLoS ONE

Article Title: Gene Silencing and Haploinsufficiency of Csk Increase Blood Pressure

doi: 10.1371/journal.pone.0146841

Figure Lengend Snippet: Decrease in blood pressure by PP2, a Src inhibitor in Csk +/- mice. (A) Csk +/- and Csk +/+ mice were i.p. injected with DMSO as control (CON) or PP2 (10 μg per kg body weight), a Src inhibitor, and blood pressure was measured 24 hours after injections. Blood pressure is expressed as the average value at each minute from 40 min to 80 min after anesthesia. (B) Blood pressure was measured in Csk +/- and Csk +/+ mice treated with DMSO or PP2 (2, and 10 μg per kg body weight) and the data is shown as the mean blood pressure for each group from 40 min to 80 min. (C) The representative blots of Csk and Src protein are shown following Western blotting in aorta of Csk +/- and Csk +/+ mice treated with DMSO or PP2 (10 μg per kg body weight). (D) Protein levels are presented as the ratio of protein band densities in Csk +/- aorta to the wild-type. (E) Human VSMC cells were transfected by Csk siRNA and then treated with DMSO or PP2 (10, 25, and 50 μM). Phosphorylated Src and Csk protein levels are shown following Western blotting.

Article Snippet: To test the effect of PP2 (Merck Millipore, 529573, Darmstadt, Germany) or PP3, a negative control inhibitor (Merck Millipore, 529574, Darmstadt, Germany), blood pressure was measured in Csk +/- and Csk +/+ mice 24 hours after i.p. injection of PP2 or PP3 at doses of 2 or 10 μg per kg body weight.

Techniques: Mouse Assay, Injection, Western Blot, Transfection