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  • 99
    Cell Signaling Technology Inc src
    Whole cell tyrosine phosphorylation (4G10) and site-specific phosphorylation of <t>Src</t> family kinases <t>(SFK),</t> LAT and PLCγ2. Platelets were stimulated with 5 µg mL −1 CRPXL for 6 min under aggregometry conditions in the presence of GR 144053 (2 µM) to prevent aggregation and facilitate solubilisation of protein. ( a ) Images from non-blinded pilot experiments show the effect of ( RS . ( b . Dashed lines (mean) and grey area (±SE) indicate background signal on the developed X-ray films. Lower panels represent total protein levels from the same samples. Concentration-responses were modelled using NONMEM 7.3 to accommodate random between experiment variability often associated with Western Blotting. Confirmation of the phosphorylation response was performed using likelihood ratio tests by comparing the following statistical hypotheses: H 0 : Max = Min and H 1 : Max ≠ Min . Results were: phospho-PLC (Y1217), P = 2 × 10 −13 (χ 2 = 65.1, df = 4); phospho-SFK (Y416), P = 7 × 10 −25 (χ 2 = 115.7, df = 3); phospho-LAT (Y200), P = 4 × 10 −10 (χ 2 = 49.8, df = 4). Evaluation of total protein levels was performed by comparing the hypotheses: H 0: Max = Min = Basal ; and H 1 : Max ≠ Min ≠ Basal ( Basal = signal when CRP = 0 µg ml −1 ). Results were: PLC, P = 0.30 (χ 2 = 6.04, df = 5); Src, P = 0.84 (χ 2 = 1.39, df = 4); LAT, P = 0.30 (χ 2 = 4.89, df = 4).
    Src, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 4039 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Santa Cruz Biotechnology c src
    Schematic model of SFK-mediated regulation of sperm AR via PKA pathway. The plasma membrane of sperm contains multiple membrane rafts where SFK are spatially and functionally associated. SFK phosphorylation/dephosphorylation are regulated by an equilibrium between C-terminal <t>Src</t> kinase (CSK) and Ca 2+ -dependent <t>PTP.</t> SFK phosphorylation/dephosphorylation is involved in the regulation of PKA activity and spontaneous AR through modulation of membrane potential.
    C Src, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 929 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc phospho src
    Schematic model of SFK-mediated regulation of sperm AR via PKA pathway. The plasma membrane of sperm contains multiple membrane rafts where SFK are spatially and functionally associated. SFK phosphorylation/dephosphorylation are regulated by an equilibrium between C-terminal <t>Src</t> kinase (CSK) and Ca 2+ -dependent <t>PTP.</t> SFK phosphorylation/dephosphorylation is involved in the regulation of PKA activity and spontaneous AR through modulation of membrane potential.
    Phospho Src, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 991 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc anti phospho src
    HSP60 associated with the phosphorylation of Erk1/2. a Analysis of mitochondrial-to-nucleus pathways in shHSP60 Panc-1 cells and control (Ctrl) cells. Cell lysates were analyzed by western blot with antibodies against P38, p-p38, <t>Src,</t> p-Src, <t>Akt1,</t> p-Akt (Thr308), p-Akt (Ser473), Erk1/2, p-Erk1/2, AMPKα, p- AMPKα, JNK, p-JNK, and actin. b–d Western blot analysis of p-Erk1/2 in the indicated cells and tissues: b shHSP60 Panc-1 cells vs. cells with rescued expression of HSP60 , c cells from PDAC cell lines (SW1990, Patu-8988, and Panc-1) vs. adjacent normal pancreatic ductal cells (hTERT-HPNE), and d fresh PDAC tissue vs. paired normal tissue. N adjacent normal tissue; T tumor tissue
    Anti Phospho Src, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 469 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti src
    HSP60 associated with the phosphorylation of Erk1/2. a Analysis of mitochondrial-to-nucleus pathways in shHSP60 Panc-1 cells and control (Ctrl) cells. Cell lysates were analyzed by western blot with antibodies against P38, p-p38, <t>Src,</t> p-Src, <t>Akt1,</t> p-Akt (Thr308), p-Akt (Ser473), Erk1/2, p-Erk1/2, AMPKα, p- AMPKα, JNK, p-JNK, and actin. b–d Western blot analysis of p-Erk1/2 in the indicated cells and tissues: b shHSP60 Panc-1 cells vs. cells with rescued expression of HSP60 , c cells from PDAC cell lines (SW1990, Patu-8988, and Panc-1) vs. adjacent normal pancreatic ductal cells (hTERT-HPNE), and d fresh PDAC tissue vs. paired normal tissue. N adjacent normal tissue; T tumor tissue
    Anti Src, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 427 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore anti src
    HSP60 associated with the phosphorylation of Erk1/2. a Analysis of mitochondrial-to-nucleus pathways in shHSP60 Panc-1 cells and control (Ctrl) cells. Cell lysates were analyzed by western blot with antibodies against P38, p-p38, <t>Src,</t> p-Src, <t>Akt1,</t> p-Akt (Thr308), p-Akt (Ser473), Erk1/2, p-Erk1/2, AMPKα, p- AMPKα, JNK, p-JNK, and actin. b–d Western blot analysis of p-Erk1/2 in the indicated cells and tissues: b shHSP60 Panc-1 cells vs. cells with rescued expression of HSP60 , c cells from PDAC cell lines (SW1990, Patu-8988, and Panc-1) vs. adjacent normal pancreatic ductal cells (hTERT-HPNE), and d fresh PDAC tissue vs. paired normal tissue. N adjacent normal tissue; T tumor tissue
    Anti Src, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 225 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    src  (Abcam)
    99
    Abcam src
    Scheme showing the proposed mechanisms for the <t>IL-1β-induced</t> alterations in membrane expression of GluA2 and GluA1 subunits of AMPA receptor in hippocampus of hyperammonemic rats. Hyperammonemia increases IL-1β, enhancing activation of IL-1 receptor. This leads to activation of <t>Src,</t> reflected in increased phosphorylation of Tyr416. a Src in turn enhances phosphorylation at Tyr1472 and membrane expression of GluN2B, which leads to activation of p38. Activated p38 binds to and reduces phosphorylation at Thr560 and activity of PKCζ, thus resulting in reduced phosphorylation at Ser880 and enhanced membrane expression of GluA2. b Src also activates PKCδ which enhances phosphorylation of GluN2B at Ser1303, reducing membrane expression of CaMKII and phosphorylation at Ser831 and membrane expression of GluA1
    Src, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Upstate Biotechnology Inc anti src
    <t>PDGF-BB-induced</t> fibroblast migration is dose-dependent and time-dependent on osteopontin; Inhibition of <t>Src</t> activation decreases PDGF-BB-induced fibroblast migration but does not block the Src association with osteopontin receptor integrin αV. ( A ) Fibroblasts were plated on osteopontin (10 μg/mg), serum-starved, wounded, treated with PDGF-BB or vehicle, and wound area was monitored for 24 hours at 37 °C. Data plotted as % of wound area covered over 24 hours relative to vehicle treated fibroblasts. ( B ) Fibroblasts were treated as Panel A and with PDGF-BB (4 ng/ml) or vehicle. ( C ) Fibroblasts were treated as Panel A with PDGF-BB (4 ng/ml), followed by Src inhibitor PP2 with indicated dose overnight, and lysed Western blotted. ( D ) Fibroblasts were wounded and treated with PDGF-BB (4 ng/ml) or vehicle, followed by PP2 (1 μM), and the monolayer wound area was monitored for 24 hours at 37 °C. ( E ) Fibroblasts were treated as in Panel A, followed by PDGF-BB (4 ng/ml) and Src inhibitor PP2 overnight. Whole cell lysates were used for coimmunoprecipitation with anti-αV, followed by Western blotted with anti-Src antibody to examine the Src-αV interaction. *Represents
    Anti Src, supplied by Upstate Biotechnology Inc, used in various techniques. Bioz Stars score: 92/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore src
    <t>PDGF-BB-induced</t> fibroblast migration is dose-dependent and time-dependent on osteopontin; Inhibition of <t>Src</t> activation decreases PDGF-BB-induced fibroblast migration but does not block the Src association with osteopontin receptor integrin αV. ( A ) Fibroblasts were plated on osteopontin (10 μg/mg), serum-starved, wounded, treated with PDGF-BB or vehicle, and wound area was monitored for 24 hours at 37 °C. Data plotted as % of wound area covered over 24 hours relative to vehicle treated fibroblasts. ( B ) Fibroblasts were treated as Panel A and with PDGF-BB (4 ng/ml) or vehicle. ( C ) Fibroblasts were treated as Panel A with PDGF-BB (4 ng/ml), followed by Src inhibitor PP2 with indicated dose overnight, and lysed Western blotted. ( D ) Fibroblasts were wounded and treated with PDGF-BB (4 ng/ml) or vehicle, followed by PP2 (1 μM), and the monolayer wound area was monitored for 24 hours at 37 °C. ( E ) Fibroblasts were treated as in Panel A, followed by PDGF-BB (4 ng/ml) and Src inhibitor PP2 overnight. Whole cell lysates were used for coimmunoprecipitation with anti-αV, followed by Western blotted with anti-Src antibody to examine the Src-αV interaction. *Represents
    Src, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 360 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    The protein encoded by this gene shares homology with Src kinase associated phosphoprotein 1 and is a substrate of Src family kinases It is an adaptor protein that is thought
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    N/A
    Non receptor protein tyrosine kinase that plays pivotal roles in numerous cellular processes such as proliferation migration and transformation In concert with PTK2B plays an important role in osteoclastic bone
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    N/A
    SRC also named as SRC1 and p60 Src belongs to the protein kinase superfamily Tyr protein kinase family and SRC subfamily It is a non receptor protein tyrosine kinase that
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    Image Search Results


    Whole cell tyrosine phosphorylation (4G10) and site-specific phosphorylation of Src family kinases (SFK), LAT and PLCγ2. Platelets were stimulated with 5 µg mL −1 CRPXL for 6 min under aggregometry conditions in the presence of GR 144053 (2 µM) to prevent aggregation and facilitate solubilisation of protein. ( a ) Images from non-blinded pilot experiments show the effect of ( RS . ( b . Dashed lines (mean) and grey area (±SE) indicate background signal on the developed X-ray films. Lower panels represent total protein levels from the same samples. Concentration-responses were modelled using NONMEM 7.3 to accommodate random between experiment variability often associated with Western Blotting. Confirmation of the phosphorylation response was performed using likelihood ratio tests by comparing the following statistical hypotheses: H 0 : Max = Min and H 1 : Max ≠ Min . Results were: phospho-PLC (Y1217), P = 2 × 10 −13 (χ 2 = 65.1, df = 4); phospho-SFK (Y416), P = 7 × 10 −25 (χ 2 = 115.7, df = 3); phospho-LAT (Y200), P = 4 × 10 −10 (χ 2 = 49.8, df = 4). Evaluation of total protein levels was performed by comparing the hypotheses: H 0: Max = Min = Basal ; and H 1 : Max ≠ Min ≠ Basal ( Basal = signal when CRP = 0 µg ml −1 ). Results were: PLC, P = 0.30 (χ 2 = 6.04, df = 5); Src, P = 0.84 (χ 2 = 1.39, df = 4); LAT, P = 0.30 (χ 2 = 4.89, df = 4).

    Journal: Scientific Reports

    Article Title: Citalopram inhibits platelet function independently of SERT-mediated 5-HT transport

    doi: 10.1038/s41598-018-21348-3

    Figure Lengend Snippet: Whole cell tyrosine phosphorylation (4G10) and site-specific phosphorylation of Src family kinases (SFK), LAT and PLCγ2. Platelets were stimulated with 5 µg mL −1 CRPXL for 6 min under aggregometry conditions in the presence of GR 144053 (2 µM) to prevent aggregation and facilitate solubilisation of protein. ( a ) Images from non-blinded pilot experiments show the effect of ( RS . ( b . Dashed lines (mean) and grey area (±SE) indicate background signal on the developed X-ray films. Lower panels represent total protein levels from the same samples. Concentration-responses were modelled using NONMEM 7.3 to accommodate random between experiment variability often associated with Western Blotting. Confirmation of the phosphorylation response was performed using likelihood ratio tests by comparing the following statistical hypotheses: H 0 : Max = Min and H 1 : Max ≠ Min . Results were: phospho-PLC (Y1217), P = 2 × 10 −13 (χ 2 = 65.1, df = 4); phospho-SFK (Y416), P = 7 × 10 −25 (χ 2 = 115.7, df = 3); phospho-LAT (Y200), P = 4 × 10 −10 (χ 2 = 49.8, df = 4). Evaluation of total protein levels was performed by comparing the hypotheses: H 0: Max = Min = Basal ; and H 1 : Max ≠ Min ≠ Basal ( Basal = signal when CRP = 0 µg ml −1 ). Results were: PLC, P = 0.30 (χ 2 = 6.04, df = 5); Src, P = 0.84 (χ 2 = 1.39, df = 4); LAT, P = 0.30 (χ 2 = 4.89, df = 4).

    Article Snippet: Antibodies against phospho-SFK (Y416) (#2101 S) , Src (#2108 S) and phospho-PLCγ2 (Y1217) (#3871) were from Cell Signalling Technology (Danvers, MA, U.S.A.).

    Techniques: Concentration Assay, Western Blot, Planar Chromatography

    TLR7 triggering leads to tyrosine kinase dependent Src K63-linked ubiquitination and TNFR-associated factor 6 (TRAF6) tyrosine phosphorylation. hTLR7-HEK293 cells, transiently transfected with plasmids coding for hemagglutinin (HA)-tagged K63-only, were pretreated or not with PP2 (20 µM) for 30 min and stimulated for 2 h with R848 (10 µM). (A) Total cell lysates were immunoprecipitated with anti-pSFKs antibody and immunoblotted using anti-HA antibody for ubiquitin detection. After stripping, the presence of p-SFKs was assessed by immunoblot analysis using specific antibodies, as control. (B) Total cell lysates were immunoprecipitated with anti-TRAF6 and immunoblotted with antibodies against pSFKs, Src, and pTyr. (C) TRAF6-specific immunoprecipitates were eluted with 2% SDS in order to disrupt the TRAF6-SFKs interaction and after dilution with PBS re-immunoprecipitated with anti-TRAF6 antibody. Results are representative of three independent experiments. Histograms on the right side of the blots show the quantification of the pulled-down proteins, calculated at least on two independent experiments. Each signal was normalized to the signal of the protein used for the pull-down and expressed as fold induction (mean ± SD) compared to untreated sample. *** P

    Journal: Frontiers in Immunology

    Article Title: Src Family Kinases Regulate Interferon Regulatory Factor 1 K63 Ubiquitination following Activation by TLR7/8 Vaccine Adjuvant in Human Monocytes and B Cells

    doi: 10.3389/fimmu.2018.00330

    Figure Lengend Snippet: TLR7 triggering leads to tyrosine kinase dependent Src K63-linked ubiquitination and TNFR-associated factor 6 (TRAF6) tyrosine phosphorylation. hTLR7-HEK293 cells, transiently transfected with plasmids coding for hemagglutinin (HA)-tagged K63-only, were pretreated or not with PP2 (20 µM) for 30 min and stimulated for 2 h with R848 (10 µM). (A) Total cell lysates were immunoprecipitated with anti-pSFKs antibody and immunoblotted using anti-HA antibody for ubiquitin detection. After stripping, the presence of p-SFKs was assessed by immunoblot analysis using specific antibodies, as control. (B) Total cell lysates were immunoprecipitated with anti-TRAF6 and immunoblotted with antibodies against pSFKs, Src, and pTyr. (C) TRAF6-specific immunoprecipitates were eluted with 2% SDS in order to disrupt the TRAF6-SFKs interaction and after dilution with PBS re-immunoprecipitated with anti-TRAF6 antibody. Results are representative of three independent experiments. Histograms on the right side of the blots show the quantification of the pulled-down proteins, calculated at least on two independent experiments. Each signal was normalized to the signal of the protein used for the pull-down and expressed as fold induction (mean ± SD) compared to untreated sample. *** P

    Article Snippet: For immunoblot and immunoprecipitation analysis, we used the following antibodies: anti-IRF-1 (cat# 8478 Cell Signaling for immunoblot and cat# sc-497 Santa Cruz Biotechnology for immunoprecipitation); anti-TRAF6 (cat# 8028 Cell Signaling); anti-Src (cat# 2108 Cell Signaling); anti-p-SFKs (Tyr416) (2101 Cell Signaling); anti-p-Tyrosine (cat# 8954 Cell Signaling); anti-HA (cat# 2367 Cell Signaling); anti-cIAP2 (cat# ab32059 Abcam), anti-β-actin (cat# ab8227 Abcam); and HRP-conjugated secondary antibodies (DAKO).

    Techniques: Transfection, Immunoprecipitation, Stripping Membranes

    Src family kinases are required for interferon regulatory factor 1 (IRF-1) protein accumulation in response to TLR7/8 stimulation in monocytes and B cells. (A,C) Immunoblot analysis of IRF-1 and p-SFKs in THP-1 (A) and primary B cells (C) pretreated with PP2 (20 µM) or DMSO alone and stimulated with R848 (10 µM) for 2 h. After stripping, filters were re-probed with an antibody against actin as loading control. Results are representative at least of three independent experiments. The histograms on the right of the blot show the results of the densitometric analysis on three independent experiments. Each signal was normalized to its respective actin signal on the same blot and expressed as fold induction (mean ± SD) compared to untreated sample. (B,D) qRT-PCR analysis of lRF-1 mRNA in THP-1 (B) and human B cells (D) pretreated with PP2 (20 µM) or DMSO alone and stimulated with R848 (10 µM) for 2 h. The relative abundance of the gene transcripts was determined on triplicate samples from at least three independent experiments using the ΔΔCt method and is expressed as the normalized fold expression (mean ± SD) compared to untreated sample. ** P

    Journal: Frontiers in Immunology

    Article Title: Src Family Kinases Regulate Interferon Regulatory Factor 1 K63 Ubiquitination following Activation by TLR7/8 Vaccine Adjuvant in Human Monocytes and B Cells

    doi: 10.3389/fimmu.2018.00330

    Figure Lengend Snippet: Src family kinases are required for interferon regulatory factor 1 (IRF-1) protein accumulation in response to TLR7/8 stimulation in monocytes and B cells. (A,C) Immunoblot analysis of IRF-1 and p-SFKs in THP-1 (A) and primary B cells (C) pretreated with PP2 (20 µM) or DMSO alone and stimulated with R848 (10 µM) for 2 h. After stripping, filters were re-probed with an antibody against actin as loading control. Results are representative at least of three independent experiments. The histograms on the right of the blot show the results of the densitometric analysis on three independent experiments. Each signal was normalized to its respective actin signal on the same blot and expressed as fold induction (mean ± SD) compared to untreated sample. (B,D) qRT-PCR analysis of lRF-1 mRNA in THP-1 (B) and human B cells (D) pretreated with PP2 (20 µM) or DMSO alone and stimulated with R848 (10 µM) for 2 h. The relative abundance of the gene transcripts was determined on triplicate samples from at least three independent experiments using the ΔΔCt method and is expressed as the normalized fold expression (mean ± SD) compared to untreated sample. ** P

    Article Snippet: For immunoblot and immunoprecipitation analysis, we used the following antibodies: anti-IRF-1 (cat# 8478 Cell Signaling for immunoblot and cat# sc-497 Santa Cruz Biotechnology for immunoprecipitation); anti-TRAF6 (cat# 8028 Cell Signaling); anti-Src (cat# 2108 Cell Signaling); anti-p-SFKs (Tyr416) (2101 Cell Signaling); anti-p-Tyrosine (cat# 8954 Cell Signaling); anti-HA (cat# 2367 Cell Signaling); anti-cIAP2 (cat# ab32059 Abcam), anti-β-actin (cat# ab8227 Abcam); and HRP-conjugated secondary antibodies (DAKO).

    Techniques: Stripping Membranes, Quantitative RT-PCR, Expressing

    EGF induces EGFR dimerization and endocytosis through c-Src/FAK-mediated cytoskeleton reorganization. ( A , D ) The dimerization level of EGFR was assessed by in situ proximity ligation assay (PLA). ( B , C ) For dimerization assay, human breast carcinoma cells were treated with increasing concentrations of EGF in the presence or absence of cytochalasin D for 1 h on ice, to allow for EGF-induced EGFR dimerization but not endocytosis. Cells were subjected to BS 3 chemical-mediated crosslinking as described in Materials and Methods. To determine the phosphorylation level of EGFR at Y1068, cells were incubated in serum-free medium (SFM) with EGF for 15 min at 37 °C. Cell extracts were assessed by Western blot analysis with the indicated antibodies. β-actin was used as a loading control. ** p

    Journal: Cancers

    Article Title: CD99–PTPN12 Axis Suppresses Actin Cytoskeleton-Mediated Dimerization of Epidermal Growth Factor Receptor

    doi: 10.3390/cancers12102895

    Figure Lengend Snippet: EGF induces EGFR dimerization and endocytosis through c-Src/FAK-mediated cytoskeleton reorganization. ( A , D ) The dimerization level of EGFR was assessed by in situ proximity ligation assay (PLA). ( B , C ) For dimerization assay, human breast carcinoma cells were treated with increasing concentrations of EGF in the presence or absence of cytochalasin D for 1 h on ice, to allow for EGF-induced EGFR dimerization but not endocytosis. Cells were subjected to BS 3 chemical-mediated crosslinking as described in Materials and Methods. To determine the phosphorylation level of EGFR at Y1068, cells were incubated in serum-free medium (SFM) with EGF for 15 min at 37 °C. Cell extracts were assessed by Western blot analysis with the indicated antibodies. β-actin was used as a loading control. ** p

    Article Snippet: Antibodies against phospho-FAK (Tyr397), FAK, phospho-EGFR (Tyr1068), PTPN12, c-Src, HER2, and Gab1 were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: In Situ, Proximity Ligation Assay, Incubation, Western Blot

    PTP4A1 enhances SRC half-life and activity in NHDF. a , b Mean ± SEM of densitometric scan expression plus representative immunoblotting for pMEK1/2 (S217/221) or pRAF (S259) (upper bands) normalized to MEK1/2 or GAPDH, respectively

    Journal: Nature Communications

    Article Title: PTP4A1 promotes TGFβ signaling and fibrosis in systemic sclerosis

    doi: 10.1038/s41467-017-01168-1

    Figure Lengend Snippet: PTP4A1 enhances SRC half-life and activity in NHDF. a , b Mean ± SEM of densitometric scan expression plus representative immunoblotting for pMEK1/2 (S217/221) or pRAF (S259) (upper bands) normalized to MEK1/2 or GAPDH, respectively

    Article Snippet: The rabbit anti-SMAD3 (catalog number 9513), rabbit anti-pSMAD3 (S423/S425, clone C25A9), rabbit anti-pERK1/2 (T202/Y204, catalog number 9101), rabbit anti-HA (clone C29F4), rabbit anti-MEK1/2 (catalog number 9122), rabbit anti-pMEK1/2 (S217/221, catalog number 9121), rabbit anti-pRAF (S259, catalog number 9421), rabbit anti-pPLCγ (Y783, catalog number 2821), rabbit anti-SRC (catalog number 2108), mouse anti-SRC (clone L4A1), rabbit anti-pSRC (Y416, catalog number 2101), rabbit anti-pSRC (Y527, catalog number 2105), and rabbit anti-GAPDH (clone 14C10) antibodies were purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Activity Assay, Expressing

    The inhibitory effect of BMP4 is not due to inhibiting PDGF-induced phosphorylation of Src or ERK in normal PASMCs from conduit pulmonary arteries. PASMCs were incubated with and without PDGF-BB (10 ng/ml) in the presence and absence of BMP4 (30 ng/ml) for 0.5 h after which p-Src, total Src, p-ERK, and total ERK were measured by Western blot analysis. A and C : representative immunoblots of p-Src, total Src, p-ERK, and total ERK. B and D : bar graph depicting the changes in p-Src and p-ERK. Results are expressed as means ± SE; n = 4. * P

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    Article Title: BMP4 inhibits PDGF-induced proliferation and collagen synthesis via PKA-mediated inhibition of calpain-2 in pulmonary artery smooth muscle cells

    doi: 10.1152/ajplung.00260.2016

    Figure Lengend Snippet: The inhibitory effect of BMP4 is not due to inhibiting PDGF-induced phosphorylation of Src or ERK in normal PASMCs from conduit pulmonary arteries. PASMCs were incubated with and without PDGF-BB (10 ng/ml) in the presence and absence of BMP4 (30 ng/ml) for 0.5 h after which p-Src, total Src, p-ERK, and total ERK were measured by Western blot analysis. A and C : representative immunoblots of p-Src, total Src, p-ERK, and total ERK. B and D : bar graph depicting the changes in p-Src and p-ERK. Results are expressed as means ± SE; n = 4. * P

    Article Snippet: Antibodies against p-ERK, total ERK, GAPDH, PKA, p-Smad 1/5, and total Smad 2/3, p-Src, and total Src (no. 4370, no. 4695, no. 2118, no. 4782, no. 9516s, no. 3102, no. 6943, and no. 2109) were from Cell Signaling Technology (Danvers, MA).

    Techniques: Incubation, Western Blot

    c-Src mediates FGFR-1 phosphorylation induced by BK/B2R system. ( A ) HUVEC and ( B ) HREC were treated with fasitibant (fas, 1 μM), then stimulated with BK (1 μM) for 15 min, and c-SRC phosphorylation was measured using western blot analysis. Results were normalized to SRC. FRSα phosphorylation was measured using western blot analysis in ( C ) HUVEC and ( D ) HREC treated with PP1 (500 nM), or SU566 (10 μM) (Src inhibitors) for 30 min, and then stimulated with BK (1 μM) for 15 min. Results were normalized to FRSα. ( E ) HUVEC were treated with PP1 (500 nM), or SU566 (10 μM) as above, and then stimulated with BK (1 μM) for 10 min. FGFR-1 was immunoprecipitated (IP), and its activation was investigated by an anti-pTYR antibody. Results were normalized to FGFR-1. The gels shown are representative of three experiments obtained with similar results. ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Bradykinin B2 Receptor Contributes to Inflammatory Responses in Human Endothelial Cells by the Transactivation of the Fibroblast Growth Factor Receptor FGFR-1

    doi: 10.3390/ijms19092638

    Figure Lengend Snippet: c-Src mediates FGFR-1 phosphorylation induced by BK/B2R system. ( A ) HUVEC and ( B ) HREC were treated with fasitibant (fas, 1 μM), then stimulated with BK (1 μM) for 15 min, and c-SRC phosphorylation was measured using western blot analysis. Results were normalized to SRC. FRSα phosphorylation was measured using western blot analysis in ( C ) HUVEC and ( D ) HREC treated with PP1 (500 nM), or SU566 (10 μM) (Src inhibitors) for 30 min, and then stimulated with BK (1 μM) for 15 min. Results were normalized to FRSα. ( E ) HUVEC were treated with PP1 (500 nM), or SU566 (10 μM) as above, and then stimulated with BK (1 μM) for 10 min. FGFR-1 was immunoprecipitated (IP), and its activation was investigated by an anti-pTYR antibody. Results were normalized to FGFR-1. The gels shown are representative of three experiments obtained with similar results. ** p

    Article Snippet: Anti-pTYR, anti-FGFR-1, anti-FGFR-2, anti-pFRSα, anti-pERK1/2, anti-ERK1/2, anti-pSTAT3, anti-STAT3, anti-pAKT, anti-AKT, anti-pSRC, and anti-SRC antibodies were from Cell Signaling (Milan, Italy).

    Techniques: Western Blot, Immunoprecipitation, Activation Assay

    Different concentrations of EGF induce Src phosphorylation at distinct residues. Western blot analysis of the effects of different concentrations of EGF on the phosphorylation levels of EGFR-Y845, Src-Y416 and Src-Y527 in MDA-MB-231 and MDA-MB-436 cells.

    Journal: PLoS ONE

    Article Title: A Switch Role of Src in the Biphasic EGF Signaling of ER-Negative Breast Cancer Cells

    doi: 10.1371/journal.pone.0041613

    Figure Lengend Snippet: Different concentrations of EGF induce Src phosphorylation at distinct residues. Western blot analysis of the effects of different concentrations of EGF on the phosphorylation levels of EGFR-Y845, Src-Y416 and Src-Y527 in MDA-MB-231 and MDA-MB-436 cells.

    Article Snippet: Phospho-EGFR and -Src antibodies, EGFR and Src antibodies, anti-phospho-p44/42 ERK (Thr202/Tyr204) (197G2) mouse monoclonal antibody (mAb) and anti-p44/42 ERK (137F5) rabbit mAb were purchased from Cell Signaling Technology (Boston, MA).

    Techniques: Western Blot, Multiple Displacement Amplification

    Src is involved in EGF induction of cyclin D1. (A). Western blot analysis of cyclin D1 expression in MDA-MB-231 and -436 cells. Cells were treated with vehicle (PBS) and EGF alone or together with the Src inhibitors PP2 and Dasatinib, the EGFR inhibitor AG1478 and PI3K inhibitor LY294002. Cell lysates were analyzed with anti-cyclin D1 antibody and anti-actin antibody was used to ensure equal loading. The experiment was repeated three times, and the representative results are shown. (B). Src inhibitors inhibit EGF induction of cyclin D1 promoter activity. ER-negative breast cancer cells were transfected with the luciferase reported plasmid cyclin D1 pl-963 that containing a luciferase gene driven by the cyclin D1 promoter. Transfected cells were treated with vehicle (PBS), 10 ng/ml or 500 ng/ml of EGF and 10 ng/ml EGF plus different inhibitors. The luciferase activities were assayed and normalized using a cytomegalovirus-driven Renilla luciferase plasmid. Columns: means of the relative luciferase activity in cells treated with vehicle that is arbitrarily set as 1.0 from four independent experiments; bars, SE. *, P

    Journal: PLoS ONE

    Article Title: A Switch Role of Src in the Biphasic EGF Signaling of ER-Negative Breast Cancer Cells

    doi: 10.1371/journal.pone.0041613

    Figure Lengend Snippet: Src is involved in EGF induction of cyclin D1. (A). Western blot analysis of cyclin D1 expression in MDA-MB-231 and -436 cells. Cells were treated with vehicle (PBS) and EGF alone or together with the Src inhibitors PP2 and Dasatinib, the EGFR inhibitor AG1478 and PI3K inhibitor LY294002. Cell lysates were analyzed with anti-cyclin D1 antibody and anti-actin antibody was used to ensure equal loading. The experiment was repeated three times, and the representative results are shown. (B). Src inhibitors inhibit EGF induction of cyclin D1 promoter activity. ER-negative breast cancer cells were transfected with the luciferase reported plasmid cyclin D1 pl-963 that containing a luciferase gene driven by the cyclin D1 promoter. Transfected cells were treated with vehicle (PBS), 10 ng/ml or 500 ng/ml of EGF and 10 ng/ml EGF plus different inhibitors. The luciferase activities were assayed and normalized using a cytomegalovirus-driven Renilla luciferase plasmid. Columns: means of the relative luciferase activity in cells treated with vehicle that is arbitrarily set as 1.0 from four independent experiments; bars, SE. *, P

    Article Snippet: Phospho-EGFR and -Src antibodies, EGFR and Src antibodies, anti-phospho-p44/42 ERK (Thr202/Tyr204) (197G2) mouse monoclonal antibody (mAb) and anti-p44/42 ERK (137F5) rabbit mAb were purchased from Cell Signaling Technology (Boston, MA).

    Techniques: Western Blot, Expressing, Multiple Displacement Amplification, Activity Assay, Transfection, Luciferase, Plasmid Preparation

    Different concentrations of EGF affect the association of EGFR and Src differently. Co-immunoprecipitation and Western blot analysis of EGFR and Src in MDA-MB-231 cells. Cells were treated with different concentrations of EGF for different time periods were lysed and the cell lysates were immunoprecipitated with pre-immune and indicated antibodies. The immunoprecipitates were blotted by indicated antibodies.

    Journal: PLoS ONE

    Article Title: A Switch Role of Src in the Biphasic EGF Signaling of ER-Negative Breast Cancer Cells

    doi: 10.1371/journal.pone.0041613

    Figure Lengend Snippet: Different concentrations of EGF affect the association of EGFR and Src differently. Co-immunoprecipitation and Western blot analysis of EGFR and Src in MDA-MB-231 cells. Cells were treated with different concentrations of EGF for different time periods were lysed and the cell lysates were immunoprecipitated with pre-immune and indicated antibodies. The immunoprecipitates were blotted by indicated antibodies.

    Article Snippet: Phospho-EGFR and -Src antibodies, EGFR and Src antibodies, anti-phospho-p44/42 ERK (Thr202/Tyr204) (197G2) mouse monoclonal antibody (mAb) and anti-p44/42 ERK (137F5) rabbit mAb were purchased from Cell Signaling Technology (Boston, MA).

    Techniques: Immunoprecipitation, Western Blot, Multiple Displacement Amplification

    Effect of CK on the activation of AKTs under Src overexpression conditions. (A and B) HEK293 cells were transfected with HA-Src for 48 h and treated with the indicated doses of CK (0–10 μM) for 24 h. (A) The levels of phosphorylated and total HA, Src, AKT1, and AKT2 proteins in the total cell lysate were determined by Western blotting analysis. (B) mRNA expression levels of AOX1 and iNOS were determined by quantitative real-time PCR. (C) HEK293 cells were treated with the indicated doses of CK (0–10 μM) for 24 h. Cell viability was determined by an MTT assay. β-actin was used as an internal control. * p

    Journal: Journal of Ginseng Research

    Article Title: Compound K, a ginsenoside metabolite, plays an antiinflammatory role in macrophages by targeting the AKT1-mediated signaling pathway

    doi: 10.1016/j.jgr.2018.10.003

    Figure Lengend Snippet: Effect of CK on the activation of AKTs under Src overexpression conditions. (A and B) HEK293 cells were transfected with HA-Src for 48 h and treated with the indicated doses of CK (0–10 μM) for 24 h. (A) The levels of phosphorylated and total HA, Src, AKT1, and AKT2 proteins in the total cell lysate were determined by Western blotting analysis. (B) mRNA expression levels of AOX1 and iNOS were determined by quantitative real-time PCR. (C) HEK293 cells were treated with the indicated doses of CK (0–10 μM) for 24 h. Cell viability was determined by an MTT assay. β-actin was used as an internal control. * p

    Article Snippet: Antibodies against Src, AKT1, AKT2, IKKα/β, and β-actin were obtained from Cell Signaling Technology (Beverly, MA, USA). qPCRBIO SyGreen Mix Lo-ROX for quantitative real-time polymerase chain reaction (PCR) was from PCR Biosystems (London, United Kingdom).

    Techniques: Activation Assay, Over Expression, Transfection, Western Blot, Expressing, Real-time Polymerase Chain Reaction, MTT Assay

    Schematic model of SFK-mediated regulation of sperm AR via PKA pathway. The plasma membrane of sperm contains multiple membrane rafts where SFK are spatially and functionally associated. SFK phosphorylation/dephosphorylation are regulated by an equilibrium between C-terminal Src kinase (CSK) and Ca 2+ -dependent PTP. SFK phosphorylation/dephosphorylation is involved in the regulation of PKA activity and spontaneous AR through modulation of membrane potential.

    Journal: PLoS ONE

    Article Title: Src family kinases-mediated negative regulation of sperm acrosome reaction in chickens (Gallus gallus domesticus)

    doi: 10.1371/journal.pone.0241181

    Figure Lengend Snippet: Schematic model of SFK-mediated regulation of sperm AR via PKA pathway. The plasma membrane of sperm contains multiple membrane rafts where SFK are spatially and functionally associated. SFK phosphorylation/dephosphorylation are regulated by an equilibrium between C-terminal Src kinase (CSK) and Ca 2+ -dependent PTP. SFK phosphorylation/dephosphorylation is involved in the regulation of PKA activity and spontaneous AR through modulation of membrane potential.

    Article Snippet: MDL-12,330A HCl (MDL), 4-hydroxyphenacyl bromide [protein tyrosine phosphatase (PTP) inhibitor], and monoclonal antibodies to c-Src (H-12) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

    Techniques: De-Phosphorylation Assay, Activity Assay

    Immunofluorescence of tumor sections from xenograft mice. Formalin-fixed, paraffin-embedded tumors were cut into 4 μm sections and then stained with anti-HER2 (red), anti-c-Src (blue), and anti-ER (green) as indicated. Magnification 200X

    Journal: BMC Cancer

    Article Title: C-Cbl reverses HER2-mediated tamoxifen resistance in human breast cancer cells

    doi: 10.1186/s12885-018-4387-5

    Figure Lengend Snippet: Immunofluorescence of tumor sections from xenograft mice. Formalin-fixed, paraffin-embedded tumors were cut into 4 μm sections and then stained with anti-HER2 (red), anti-c-Src (blue), and anti-ER (green) as indicated. Magnification 200X

    Article Snippet: The sections were permeabilized with 0.2% Triton X-100 for 5 min, blocked with 5% bovine serum albumin for 1 h and then incubated with phycoerythrin-labeled anti-HER2 (Sc-33684PE), anti-c-Src (SC8056), and anti-ER (SC543) antibodies (all purchased from Santa Cruz) overnight at 4 °C.

    Techniques: Immunofluorescence, Mouse Assay, Formalin-fixed Paraffin-Embedded, Staining

    Chromatin immunoprecipitation assay and Western blotting analysis of c-Myc, iNOS, Cyclin D1, and VEGF expression in Panc-1 and Colo-357 cells. (A), Agarose gel electrophoresis of the Polymerase Chain Reaction (PCR)-amplified c-Myc gene fragment from the chromatin DNA precipitated with antibody against EGFR, Src, or Stat3, or with the non-specific IgG; and (B and C), Immunoblotting analysis of whole-cell lysates probing for EGFR or Src (B(i) and C(i)) or c-Myc, iNOS, Cyclin D1 or VEGF (B(ii) and C(ii)), and the effects of siRNA knockdown of EGFR (EGFR siRNA), Src (Src siRNA) or control (con) siRNA, or S3I-201 or Das). Bands corresponding to proteins or c-Myc gene in gel are shown; M, molecular weight marker, EGFR/Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody. Data are representative of 3 independent studies, and values are mean and s.d of 3 independent studies; * p -

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Chromatin immunoprecipitation assay and Western blotting analysis of c-Myc, iNOS, Cyclin D1, and VEGF expression in Panc-1 and Colo-357 cells. (A), Agarose gel electrophoresis of the Polymerase Chain Reaction (PCR)-amplified c-Myc gene fragment from the chromatin DNA precipitated with antibody against EGFR, Src, or Stat3, or with the non-specific IgG; and (B and C), Immunoblotting analysis of whole-cell lysates probing for EGFR or Src (B(i) and C(i)) or c-Myc, iNOS, Cyclin D1 or VEGF (B(ii) and C(ii)), and the effects of siRNA knockdown of EGFR (EGFR siRNA), Src (Src siRNA) or control (con) siRNA, or S3I-201 or Das). Bands corresponding to proteins or c-Myc gene in gel are shown; M, molecular weight marker, EGFR/Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody. Data are representative of 3 independent studies, and values are mean and s.d of 3 independent studies; * p -

    Article Snippet: Then the proteins were eluted with freshly prepared elution buffer (1% SDS, 100 mM NaHCO3 ) and subjected to the second immunoprecipitation by incubating with anti-Src antibody or IgG (Santa Cruz).

    Techniques: Chromatin Immunoprecipitation, Western Blot, Expressing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Molecular Weight, Marker, Immunoprecipitation

    Co-immunoprecipitation with immunoblotting analysis of the effects of modulation of EGFR, Src and Stat3 on the nuclear EGFR, Src and Stat3 complex. (A, B, and C) Immunoblotting analyses of immunecomplexes of Stat3 (IP:Stat3), EGFR (IP:EGFR), or Src (IP:Src) prepared from nuclear extracts of Panc-1 cells untransfected or transfected with Src siRNA, EGFR siRNA, or control (con) siRNA (A), or treated with or without the EGFR inhibitor (ZD1839, ZD), Src inhibitor (Dasatinib, Das), or the Stat3 inhibitor (S3I-201) for 1 or 24 h (B), or from nuclear extracts pre-incubated for 2 h with or without 100 µM pY1068, pY1086, or SPI peptide (C) and probing for EGFR, Src, Stat3; or (D) immunoblotting analysis of nuclear extracts prepared from Panc-1 cells treated or untreated with phenylarsine oxide (PAO) and probing for Src, Stat3, EGFR. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of lysate or nuclear extract used in the assay; Data are representative of 3 independent studies.

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Co-immunoprecipitation with immunoblotting analysis of the effects of modulation of EGFR, Src and Stat3 on the nuclear EGFR, Src and Stat3 complex. (A, B, and C) Immunoblotting analyses of immunecomplexes of Stat3 (IP:Stat3), EGFR (IP:EGFR), or Src (IP:Src) prepared from nuclear extracts of Panc-1 cells untransfected or transfected with Src siRNA, EGFR siRNA, or control (con) siRNA (A), or treated with or without the EGFR inhibitor (ZD1839, ZD), Src inhibitor (Dasatinib, Das), or the Stat3 inhibitor (S3I-201) for 1 or 24 h (B), or from nuclear extracts pre-incubated for 2 h with or without 100 µM pY1068, pY1086, or SPI peptide (C) and probing for EGFR, Src, Stat3; or (D) immunoblotting analysis of nuclear extracts prepared from Panc-1 cells treated or untreated with phenylarsine oxide (PAO) and probing for Src, Stat3, EGFR. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of lysate or nuclear extract used in the assay; Data are representative of 3 independent studies.

    Article Snippet: Then the proteins were eluted with freshly prepared elution buffer (1% SDS, 100 mM NaHCO3 ) and subjected to the second immunoprecipitation by incubating with anti-Src antibody or IgG (Santa Cruz).

    Techniques: Immunoprecipitation, Transfection, Incubation

    Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 complex in the nucleus and the sub-cellular distribution of EGFR, Src and Stat3. (A and B) Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), Stat3 (IP:Stat3), EGFR/Src (IP:EGFR/IP:Src), or of non-specific IgG non-immuneprecpitate prepared from nuclear extracts of Panc-1 or Colo-357 cells and probing for Stat3, EGFR, Src, or the Tata-binding protein (TBP); and (C), immunoblotting analysis of membrane (mem) and cytosolic (cyto) fractions and of nuclear (nuc) extracts from Panc-1 cells probing for (i) EGFR, (ii) Stat3 and (iii) Src. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of nuclear extract used in the assay; IP:EGFR/IP:Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody; Data are representative of 3 independent studies.

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 complex in the nucleus and the sub-cellular distribution of EGFR, Src and Stat3. (A and B) Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), Stat3 (IP:Stat3), EGFR/Src (IP:EGFR/IP:Src), or of non-specific IgG non-immuneprecpitate prepared from nuclear extracts of Panc-1 or Colo-357 cells and probing for Stat3, EGFR, Src, or the Tata-binding protein (TBP); and (C), immunoblotting analysis of membrane (mem) and cytosolic (cyto) fractions and of nuclear (nuc) extracts from Panc-1 cells probing for (i) EGFR, (ii) Stat3 and (iii) Src. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of nuclear extract used in the assay; IP:EGFR/IP:Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody; Data are representative of 3 independent studies.

    Article Snippet: Then the proteins were eluted with freshly prepared elution buffer (1% SDS, 100 mM NaHCO3 ) and subjected to the second immunoprecipitation by incubating with anti-Src antibody or IgG (Santa Cruz).

    Techniques: Immunoprecipitation, Binding Assay

    Studies of protein complex and protein binding partners using the Detection and Analysis through Nanoparticle Sizing technology. (A) Kinetic binding assay of EGFR-gold nanoparticle (GNP) probe (or mouse IgG1-GNP probe as negative control) binding to (i) EGFR protein and its complex from Panc-1 nuclear extracts, and the (ii) inhibitory effect of the mouse monoclonal anti-EGFR antibody on the EGFR-GNP probe binding to the EGFR protein; and (B) Protein complex binding partner analysis whereby the polyclonal anti-Stat3, anti-Src or anti-EGFR antibody or the non-specific rabbit IgG (negative control) is added to the assay solution prepared from the (i) non-specific mouse IgG1-GNP probe (negative control), or (ii) anti-EGFR-GNP probe; Data are representative of 4 independent studies.

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Studies of protein complex and protein binding partners using the Detection and Analysis through Nanoparticle Sizing technology. (A) Kinetic binding assay of EGFR-gold nanoparticle (GNP) probe (or mouse IgG1-GNP probe as negative control) binding to (i) EGFR protein and its complex from Panc-1 nuclear extracts, and the (ii) inhibitory effect of the mouse monoclonal anti-EGFR antibody on the EGFR-GNP probe binding to the EGFR protein; and (B) Protein complex binding partner analysis whereby the polyclonal anti-Stat3, anti-Src or anti-EGFR antibody or the non-specific rabbit IgG (negative control) is added to the assay solution prepared from the (i) non-specific mouse IgG1-GNP probe (negative control), or (ii) anti-EGFR-GNP probe; Data are representative of 4 independent studies.

    Article Snippet: Then the proteins were eluted with freshly prepared elution buffer (1% SDS, 100 mM NaHCO3 ) and subjected to the second immunoprecipitation by incubating with anti-Src antibody or IgG (Santa Cruz).

    Techniques: Protein Binding, Binding Assay, Negative Control

    Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 association in Panc-1 and Colo-357 cells. Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), and Stat3 (IP:Stat3), or of non-specific IgG non-immunoprecipitate prepared from whole-cell lysates of Panc-1 or Colo-357 cells untransfected (A and B) or transfected with EGFR siRNA, Src siRNA, or control (con) siRNA (C) and probing for Src, Stat3 and EGFR in the absence (A and C) or presence (B) of Stat3 blocking peptide (Stat3 BP), Src blocking peptide (Src BP) or EGFR blocking peptide (EGFR BP). Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of lysate used in the assay; Data are representative of 3 independent studies.

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 association in Panc-1 and Colo-357 cells. Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), and Stat3 (IP:Stat3), or of non-specific IgG non-immunoprecipitate prepared from whole-cell lysates of Panc-1 or Colo-357 cells untransfected (A and B) or transfected with EGFR siRNA, Src siRNA, or control (con) siRNA (C) and probing for Src, Stat3 and EGFR in the absence (A and C) or presence (B) of Stat3 blocking peptide (Stat3 BP), Src blocking peptide (Src BP) or EGFR blocking peptide (EGFR BP). Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of lysate used in the assay; Data are representative of 3 independent studies.

    Article Snippet: Then the proteins were eluted with freshly prepared elution buffer (1% SDS, 100 mM NaHCO3 ) and subjected to the second immunoprecipitation by incubating with anti-Src antibody or IgG (Santa Cruz).

    Techniques: Immunoprecipitation, Transfection, Blocking Assay

    HSP60 associated with the phosphorylation of Erk1/2. a Analysis of mitochondrial-to-nucleus pathways in shHSP60 Panc-1 cells and control (Ctrl) cells. Cell lysates were analyzed by western blot with antibodies against P38, p-p38, Src, p-Src, Akt1, p-Akt (Thr308), p-Akt (Ser473), Erk1/2, p-Erk1/2, AMPKα, p- AMPKα, JNK, p-JNK, and actin. b–d Western blot analysis of p-Erk1/2 in the indicated cells and tissues: b shHSP60 Panc-1 cells vs. cells with rescued expression of HSP60 , c cells from PDAC cell lines (SW1990, Patu-8988, and Panc-1) vs. adjacent normal pancreatic ductal cells (hTERT-HPNE), and d fresh PDAC tissue vs. paired normal tissue. N adjacent normal tissue; T tumor tissue

    Journal: Cell Death & Disease

    Article Title: Oncogenic HSP60 regulates mitochondrial oxidative phosphorylation to support Erk1/2 activation during pancreatic cancer cell growth

    doi: 10.1038/s41419-017-0196-z

    Figure Lengend Snippet: HSP60 associated with the phosphorylation of Erk1/2. a Analysis of mitochondrial-to-nucleus pathways in shHSP60 Panc-1 cells and control (Ctrl) cells. Cell lysates were analyzed by western blot with antibodies against P38, p-p38, Src, p-Src, Akt1, p-Akt (Thr308), p-Akt (Ser473), Erk1/2, p-Erk1/2, AMPKα, p- AMPKα, JNK, p-JNK, and actin. b–d Western blot analysis of p-Erk1/2 in the indicated cells and tissues: b shHSP60 Panc-1 cells vs. cells with rescued expression of HSP60 , c cells from PDAC cell lines (SW1990, Patu-8988, and Panc-1) vs. adjacent normal pancreatic ductal cells (hTERT-HPNE), and d fresh PDAC tissue vs. paired normal tissue. N adjacent normal tissue; T tumor tissue

    Article Snippet: Proteins were probed with anti-Hsp60 (sc-376261; 1:1000; Santa Cruz Biotechnology), anti-ERK1/2 (#9102; 1:1000; Cell Signaling Technology), anti-phospho-ERK (Thr202/Tyr204) (#9101; 1:1000; Cell Signaling Technology), anti-AMPKα (#2532; 1:1000; Cell Signaling Technology), anti-phospho-AMPKα (Thr172) (#2535; 1:1000; Cell Signaling Technology), anti-P38 (#9212; 1:1000; Cell Signaling Technology), anti-phospho-P38 (Thr389) (#9211; 1:1000; Cell Signaling Technology), anti-Src (#2109; 1:1000; Cell Signaling Technology), anti-phospho-Src (#2105; 1:1000; Cell Signaling Technology), anti-AKT1 (#2938; 1:1000; Cell Signaling Technology), anti-phospho-AKT (Ser473) (#12694; 1:1000; Cell Signaling Technology), anti-phospho-AKT (Thr308) (#5106; 1:1000; Cell Signaling Technology), anti-JNK (#9252; 1:1000; Cell Signaling Technology), anti-phospho-JNK (#4668; 1:1000; Cell Signaling Technology), anti-NDUFA13 (ab110240; 1:1000; Abcam, Cambridge, MA, USA), anti-SDHA (ab14715; 1:1000; Abcam), anti-core2 (MS304; 1:1000; Abcam), anti-COXI (MS404; 1:1000; Abcam), anti-ATP synthase subunit alpha (ab14748; 1:1000; Abcam), anti-β-actin (sc-47778; 1:5000; Santa Cruz Biotechnology) or anti-VDAC (#4661; 1:1000; Cell Signaling Technology) antibodies, and then incubated with a horseradish peroxidase-conjugated anti-rabbit/mouse IgG (#7074 / #7076; 1:2000; Cell Signaling Technology) secondary antibody.

    Techniques: Western Blot, Expressing

    Scheme showing the proposed mechanisms for the IL-1β-induced alterations in membrane expression of GluA2 and GluA1 subunits of AMPA receptor in hippocampus of hyperammonemic rats. Hyperammonemia increases IL-1β, enhancing activation of IL-1 receptor. This leads to activation of Src, reflected in increased phosphorylation of Tyr416. a Src in turn enhances phosphorylation at Tyr1472 and membrane expression of GluN2B, which leads to activation of p38. Activated p38 binds to and reduces phosphorylation at Thr560 and activity of PKCζ, thus resulting in reduced phosphorylation at Ser880 and enhanced membrane expression of GluA2. b Src also activates PKCδ which enhances phosphorylation of GluN2B at Ser1303, reducing membrane expression of CaMKII and phosphorylation at Ser831 and membrane expression of GluA1

    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: Scheme showing the proposed mechanisms for the IL-1β-induced alterations in membrane expression of GluA2 and GluA1 subunits of AMPA receptor in hippocampus of hyperammonemic rats. Hyperammonemia increases IL-1β, enhancing activation of IL-1 receptor. This leads to activation of Src, reflected in increased phosphorylation of Tyr416. a Src in turn enhances phosphorylation at Tyr1472 and membrane expression of GluN2B, which leads to activation of p38. Activated p38 binds to and reduces phosphorylation at Thr560 and activity of PKCζ, thus resulting in reduced phosphorylation at Ser880 and enhanced membrane expression of GluA2. b Src also activates PKCδ which enhances phosphorylation of GluN2B at Ser1303, reducing membrane expression of CaMKII and phosphorylation at Ser831 and membrane expression of GluA1

    Article Snippet: Samples were subjected to immunoblotting as in Felipo et al. [ ], using antibodies against IL-1β (1:500, cat# AF-501-NA) from R & D Systems; Src (1:1000, cat# ab47405), Src phosphorylated at Tyr416 (1:1000, cat# ab40660), GluN2B phosphorylated at Ser1303 (1:1000, cat# ab81271), GluA2 phosphorylated at Ser880 (1:2000, cat# ab52180), and PKCζ phosphorylated at Thr560 (1:1000, cat# ab59412) from Abcam; GluN2B (1:1000, cat# 06-600), GluN2B phosphorylated at Tyr1472 (1:1000, cat# AB5403), GluA1 (1:1000, cat# 04-855), GluA1 phosphorylated at Ser831 (1:1000, cat# 04-823), and GluA2 (1:2000, cat# AB1768) from Millipore (Darmstadt, Germany); p38 (1:1000, cat# 9212) and p38 phosphorylated at Thr180/Tyr182 (1:500, cat# 9211) from Cell Signaling (Leiden, Netherlands); and PKCζ (1:2000, cat# sc-17,781) from Santa Cruz (Dallas, TX).

    Techniques: Expressing, Activation Assay, Activity Assay

    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

    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: Samples were subjected to immunoblotting as in Felipo et al. [ ], using antibodies against IL-1β (1:500, cat# AF-501-NA) from R & D Systems; Src (1:1000, cat# ab47405), Src phosphorylated at Tyr416 (1:1000, cat# ab40660), GluN2B phosphorylated at Ser1303 (1:1000, cat# ab81271), GluA2 phosphorylated at Ser880 (1:2000, cat# ab52180), and PKCζ phosphorylated at Thr560 (1:1000, cat# ab59412) from Abcam; GluN2B (1:1000, cat# 06-600), GluN2B phosphorylated at Tyr1472 (1:1000, cat# AB5403), GluA1 (1:1000, cat# 04-855), GluA1 phosphorylated at Ser831 (1:1000, cat# 04-823), and GluA2 (1:2000, cat# AB1768) from Millipore (Darmstadt, Germany); p38 (1:1000, cat# 9212) and p38 phosphorylated at Thr180/Tyr182 (1:500, cat# 9211) from Cell Signaling (Leiden, Netherlands); and PKCζ (1:2000, cat# sc-17,781) from Santa Cruz (Dallas, TX).

    Techniques: Activation Assay, Expressing

    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

    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: Samples were subjected to immunoblotting as in Felipo et al. [ ], using antibodies against IL-1β (1:500, cat# AF-501-NA) from R & D Systems; Src (1:1000, cat# ab47405), Src phosphorylated at Tyr416 (1:1000, cat# ab40660), GluN2B phosphorylated at Ser1303 (1:1000, cat# ab81271), GluA2 phosphorylated at Ser880 (1:2000, cat# ab52180), and PKCζ phosphorylated at Thr560 (1:1000, cat# ab59412) from Abcam; GluN2B (1:1000, cat# 06-600), GluN2B phosphorylated at Tyr1472 (1:1000, cat# AB5403), GluA1 (1:1000, cat# 04-855), GluA1 phosphorylated at Ser831 (1:1000, cat# 04-823), and GluA2 (1:2000, cat# AB1768) from Millipore (Darmstadt, Germany); p38 (1:1000, cat# 9212) and p38 phosphorylated at Thr180/Tyr182 (1:500, cat# 9211) from Cell Signaling (Leiden, Netherlands); and PKCζ (1:2000, cat# sc-17,781) from Santa Cruz (Dallas, TX).

    Techniques: Activation Assay

    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

    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: Samples were subjected to immunoblotting as in Felipo et al. [ ], using antibodies against IL-1β (1:500, cat# AF-501-NA) from R & D Systems; Src (1:1000, cat# ab47405), Src phosphorylated at Tyr416 (1:1000, cat# ab40660), GluN2B phosphorylated at Ser1303 (1:1000, cat# ab81271), GluA2 phosphorylated at Ser880 (1:2000, cat# ab52180), and PKCζ phosphorylated at Thr560 (1:1000, cat# ab59412) from Abcam; GluN2B (1:1000, cat# 06-600), GluN2B phosphorylated at Tyr1472 (1:1000, cat# AB5403), GluA1 (1:1000, cat# 04-855), GluA1 phosphorylated at Ser831 (1:1000, cat# 04-823), and GluA2 (1:2000, cat# AB1768) from Millipore (Darmstadt, Germany); p38 (1:1000, cat# 9212) and p38 phosphorylated at Thr180/Tyr182 (1:500, cat# 9211) from Cell Signaling (Leiden, Netherlands); and PKCζ (1:2000, cat# sc-17,781) from Santa Cruz (Dallas, TX).

    Techniques: Activation Assay, Expressing

    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

    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: Samples were subjected to immunoblotting as in Felipo et al. [ ], using antibodies against IL-1β (1:500, cat# AF-501-NA) from R & D Systems; Src (1:1000, cat# ab47405), Src phosphorylated at Tyr416 (1:1000, cat# ab40660), GluN2B phosphorylated at Ser1303 (1:1000, cat# ab81271), GluA2 phosphorylated at Ser880 (1:2000, cat# ab52180), and PKCζ phosphorylated at Thr560 (1:1000, cat# ab59412) from Abcam; GluN2B (1:1000, cat# 06-600), GluN2B phosphorylated at Tyr1472 (1:1000, cat# AB5403), GluA1 (1:1000, cat# 04-855), GluA1 phosphorylated at Ser831 (1:1000, cat# 04-823), and GluA2 (1:2000, cat# AB1768) from Millipore (Darmstadt, Germany); p38 (1:1000, cat# 9212) and p38 phosphorylated at Thr180/Tyr182 (1:500, cat# 9211) from Cell Signaling (Leiden, Netherlands); and PKCζ (1:2000, cat# sc-17,781) from Santa Cruz (Dallas, TX).

    Techniques: Activation Assay, Expressing

    PDGF-BB-induced fibroblast migration is dose-dependent and time-dependent on osteopontin; Inhibition of Src activation decreases PDGF-BB-induced fibroblast migration but does not block the Src association with osteopontin receptor integrin αV. ( A ) Fibroblasts were plated on osteopontin (10 μg/mg), serum-starved, wounded, treated with PDGF-BB or vehicle, and wound area was monitored for 24 hours at 37 °C. Data plotted as % of wound area covered over 24 hours relative to vehicle treated fibroblasts. ( B ) Fibroblasts were treated as Panel A and with PDGF-BB (4 ng/ml) or vehicle. ( C ) Fibroblasts were treated as Panel A with PDGF-BB (4 ng/ml), followed by Src inhibitor PP2 with indicated dose overnight, and lysed Western blotted. ( D ) Fibroblasts were wounded and treated with PDGF-BB (4 ng/ml) or vehicle, followed by PP2 (1 μM), and the monolayer wound area was monitored for 24 hours at 37 °C. ( E ) Fibroblasts were treated as in Panel A, followed by PDGF-BB (4 ng/ml) and Src inhibitor PP2 overnight. Whole cell lysates were used for coimmunoprecipitation with anti-αV, followed by Western blotted with anti-Src antibody to examine the Src-αV interaction. *Represents

    Journal: Scientific Reports

    Article Title: Interaction of Src and Alpha-V Integrin Regulates Fibroblast Migration and Modulates Lung Fibrosis in A Preclinical Model of Lung Fibrosis

    doi: 10.1038/srep46357

    Figure Lengend Snippet: PDGF-BB-induced fibroblast migration is dose-dependent and time-dependent on osteopontin; Inhibition of Src activation decreases PDGF-BB-induced fibroblast migration but does not block the Src association with osteopontin receptor integrin αV. ( A ) Fibroblasts were plated on osteopontin (10 μg/mg), serum-starved, wounded, treated with PDGF-BB or vehicle, and wound area was monitored for 24 hours at 37 °C. Data plotted as % of wound area covered over 24 hours relative to vehicle treated fibroblasts. ( B ) Fibroblasts were treated as Panel A and with PDGF-BB (4 ng/ml) or vehicle. ( C ) Fibroblasts were treated as Panel A with PDGF-BB (4 ng/ml), followed by Src inhibitor PP2 with indicated dose overnight, and lysed Western blotted. ( D ) Fibroblasts were wounded and treated with PDGF-BB (4 ng/ml) or vehicle, followed by PP2 (1 μM), and the monolayer wound area was monitored for 24 hours at 37 °C. ( E ) Fibroblasts were treated as in Panel A, followed by PDGF-BB (4 ng/ml) and Src inhibitor PP2 overnight. Whole cell lysates were used for coimmunoprecipitation with anti-αV, followed by Western blotted with anti-Src antibody to examine the Src-αV interaction. *Represents

    Article Snippet: Reagents The following reagents and antibodies were commercially purchased: PDGF-BB (R & D Systems), anti-phospho-Src [pY416] (Cell Signaling), anti-Src (Upstate Biotechnology), mouse IgG (Molecular Probes), beta-1 (β1), β6, αVβ3, αVβ5, αV, and β8 (Abcam), pro-collagen, fibronectin (FN), anti-cyclin D1, and beta-actin (Santa Cruz Biotechnology), PP2, anti-α-SMA mouse IgG, fibronectin (FN), bleomycin, and other chemicals (Sigma).

    Techniques: Migration, Inhibition, Activation Assay, Blocking Assay, Western Blot

    Src is activated in a dose-dependent and time-dependent manner, and directly associated with integrin αV in response to PDGF-BB treatment in fibroblasts. ( A ) Fibroblasts were planted on osteopontin (10 μg/mg), serum starved, treated with PDGF-BB for overnight, and lysed for Western blot. ( B ) Densitometry of Src activation. pY416 of Src levels were normalized to total Src levels. Data were represented as the percentage of Src activation relative to that in highest dose (8 ng/ml, set as 100%). ( C ) Cell lysates were Western blotted with indicated antibodies. ( D ) Densitometry of Src activation from Panel C. pY416 of Src levels were normalized to total Src levels. Data were represented as the percentage of Src activation relative to that in longest time point (24 hours, set as 100. ( E ) Whole cell lysates were used for coimmunoprecipitation with anti- αV IgG, followed by Western blotted with anti-Src antibody to examine the Src-αV interaction. ( F ) Densitometry of Src-αV association from Panel E. Data were represented as the percentage of association elative to that in longest time point (24 hours, set as 100%). *Represents

    Journal: Scientific Reports

    Article Title: Interaction of Src and Alpha-V Integrin Regulates Fibroblast Migration and Modulates Lung Fibrosis in A Preclinical Model of Lung Fibrosis

    doi: 10.1038/srep46357

    Figure Lengend Snippet: Src is activated in a dose-dependent and time-dependent manner, and directly associated with integrin αV in response to PDGF-BB treatment in fibroblasts. ( A ) Fibroblasts were planted on osteopontin (10 μg/mg), serum starved, treated with PDGF-BB for overnight, and lysed for Western blot. ( B ) Densitometry of Src activation. pY416 of Src levels were normalized to total Src levels. Data were represented as the percentage of Src activation relative to that in highest dose (8 ng/ml, set as 100%). ( C ) Cell lysates were Western blotted with indicated antibodies. ( D ) Densitometry of Src activation from Panel C. pY416 of Src levels were normalized to total Src levels. Data were represented as the percentage of Src activation relative to that in longest time point (24 hours, set as 100. ( E ) Whole cell lysates were used for coimmunoprecipitation with anti- αV IgG, followed by Western blotted with anti-Src antibody to examine the Src-αV interaction. ( F ) Densitometry of Src-αV association from Panel E. Data were represented as the percentage of association elative to that in longest time point (24 hours, set as 100%). *Represents

    Article Snippet: Reagents The following reagents and antibodies were commercially purchased: PDGF-BB (R & D Systems), anti-phospho-Src [pY416] (Cell Signaling), anti-Src (Upstate Biotechnology), mouse IgG (Molecular Probes), beta-1 (β1), β6, αVβ3, αVβ5, αV, and β8 (Abcam), pro-collagen, fibronectin (FN), anti-cyclin D1, and beta-actin (Santa Cruz Biotechnology), PP2, anti-α-SMA mouse IgG, fibronectin (FN), bleomycin, and other chemicals (Sigma).

    Techniques: Western Blot, Activation Assay

    Integrins αVβ5 and αVβ3 are main integrin receptors contributing to Src-mediated fibroblast migration and Src activation on osteopontin. ( A ) Fibroblasts were planted on osteopontin (OPN, 10 μg/mg), serum starved, wounded as in Panel A, treated with PDGF-BB (4 ng/ml), followed by αV integrin blocking antibody. The monolayer wound area was monitored for 24 hours at 37 °C. Data plotted as % of wound area covered over 24 hours relative to control IgG treated fibroblasts (bar 1, defined as 100%). ( B ) Fibroblasts were treated as in Panel A and with PDGF-BB (4 ng/ml), followed by indicated integrin blocking antibodies or control mouse IgG. The monolayer wound area was monitored for 24 hours at 37 °C. Data were plotted as % of wound area covered over 24 hours relative to control IgG treated fibroblasts. ( C ) Fibroblasts were treated as in Panel A and lysed for Src activation. ( D ) Fibroblasts were treated as in Panel A and lysed. Whole cell lysates were used for coimmunoprecipitation with anti-αV IgG, followed by Western blotted with anti-Src antibody to examine the Src-αV interaction. *Represents

    Journal: Scientific Reports

    Article Title: Interaction of Src and Alpha-V Integrin Regulates Fibroblast Migration and Modulates Lung Fibrosis in A Preclinical Model of Lung Fibrosis

    doi: 10.1038/srep46357

    Figure Lengend Snippet: Integrins αVβ5 and αVβ3 are main integrin receptors contributing to Src-mediated fibroblast migration and Src activation on osteopontin. ( A ) Fibroblasts were planted on osteopontin (OPN, 10 μg/mg), serum starved, wounded as in Panel A, treated with PDGF-BB (4 ng/ml), followed by αV integrin blocking antibody. The monolayer wound area was monitored for 24 hours at 37 °C. Data plotted as % of wound area covered over 24 hours relative to control IgG treated fibroblasts (bar 1, defined as 100%). ( B ) Fibroblasts were treated as in Panel A and with PDGF-BB (4 ng/ml), followed by indicated integrin blocking antibodies or control mouse IgG. The monolayer wound area was monitored for 24 hours at 37 °C. Data were plotted as % of wound area covered over 24 hours relative to control IgG treated fibroblasts. ( C ) Fibroblasts were treated as in Panel A and lysed for Src activation. ( D ) Fibroblasts were treated as in Panel A and lysed. Whole cell lysates were used for coimmunoprecipitation with anti-αV IgG, followed by Western blotted with anti-Src antibody to examine the Src-αV interaction. *Represents

    Article Snippet: Reagents The following reagents and antibodies were commercially purchased: PDGF-BB (R & D Systems), anti-phospho-Src [pY416] (Cell Signaling), anti-Src (Upstate Biotechnology), mouse IgG (Molecular Probes), beta-1 (β1), β6, αVβ3, αVβ5, αV, and β8 (Abcam), pro-collagen, fibronectin (FN), anti-cyclin D1, and beta-actin (Santa Cruz Biotechnology), PP2, anti-α-SMA mouse IgG, fibronectin (FN), bleomycin, and other chemicals (Sigma).

    Techniques: Migration, Activation Assay, Blocking Assay, Western Blot

    Src inhibitor protects lung fibrosis in bleomycin-challenged mice. ( A ) Mice were instilled with saline (Sal) or bleomycin (Bleo) and followed by daily treatment of PP2 (50 mg/kg, i.p. injection) or vehicle. Lung tissues at day 21 were HE stained (200×). ( B ) The severity of lung fibrosis was examined morphometrically and represented by Ashcroft Score. ( C ) Lung hydroxyproline level was measured and represented as % hydroxyproline normalized to that in vehicle-challenged mice. ( D ) Lung tissue lysates were Western blotted. ( E ) Densitometry of Src activation from Panel D. pY416 of Src levels were normalized to total Src levels. Data were represented as the fold of Src activation relative to that in the control vehicle and saline treated group. ( F ) Densitometry of total Src protein from Panel D. Src levels were normalized to G3PDH levels. Data were represented as the fold of Src relative to that in the control vehicle and saline treated group. Per experimental group had 8–12 mice. Data were represented as mean + SE. * represents p

    Journal: Scientific Reports

    Article Title: Interaction of Src and Alpha-V Integrin Regulates Fibroblast Migration and Modulates Lung Fibrosis in A Preclinical Model of Lung Fibrosis

    doi: 10.1038/srep46357

    Figure Lengend Snippet: Src inhibitor protects lung fibrosis in bleomycin-challenged mice. ( A ) Mice were instilled with saline (Sal) or bleomycin (Bleo) and followed by daily treatment of PP2 (50 mg/kg, i.p. injection) or vehicle. Lung tissues at day 21 were HE stained (200×). ( B ) The severity of lung fibrosis was examined morphometrically and represented by Ashcroft Score. ( C ) Lung hydroxyproline level was measured and represented as % hydroxyproline normalized to that in vehicle-challenged mice. ( D ) Lung tissue lysates were Western blotted. ( E ) Densitometry of Src activation from Panel D. pY416 of Src levels were normalized to total Src levels. Data were represented as the fold of Src activation relative to that in the control vehicle and saline treated group. ( F ) Densitometry of total Src protein from Panel D. Src levels were normalized to G3PDH levels. Data were represented as the fold of Src relative to that in the control vehicle and saline treated group. Per experimental group had 8–12 mice. Data were represented as mean + SE. * represents p

    Article Snippet: Reagents The following reagents and antibodies were commercially purchased: PDGF-BB (R & D Systems), anti-phospho-Src [pY416] (Cell Signaling), anti-Src (Upstate Biotechnology), mouse IgG (Molecular Probes), beta-1 (β1), β6, αVβ3, αVβ5, αV, and β8 (Abcam), pro-collagen, fibronectin (FN), anti-cyclin D1, and beta-actin (Santa Cruz Biotechnology), PP2, anti-α-SMA mouse IgG, fibronectin (FN), bleomycin, and other chemicals (Sigma).

    Techniques: Mouse Assay, Injection, Staining, Western Blot, Activation Assay