vegfr2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc vegfr2
    The effects of irradiated conditioned medium (IR-CM) on activation of kinases in glioma cells . After 16 h incubation of GBM glioma cells with IR-CM ± monoclonal anti-human VEGF 165 antibody (R&D System), cells were harvested, processed to make lysates and the supernatant was collected. Equal quantities of protein were separated by SDS/PAGE and transferred to PVDF membrane. The membranes were blocked with 5% dried non-fat milk for 1 h at room temperature and incubated with the desired primary antibody diluted 1:1000 in blocking buffer overnight at 4 C. Membranes were probed with antibodies to phospho <t>VEGFR2</t> Y996 , phospho VEGFR2 Y1056 , Src Y461 , phospho FAK Y861 and phospho FAK Y925 . Primary antibody incubation was followed by incubation with a horseradish peroxidase-conjugated secondary antibody in blocking buffer for 2 h at room temperature. Proteins were visualized with electrochemiluminescence detection reagents and detected by autoradiography. ( A ) IR-CM effect on phosphorylation of VEGFR2 in U251 glioma cells. ( B ) IR-CM with/without VEGF antibody (10 μg/ml) effect on the phosphorylation of Src and FAK in U251 glioma cells. Blots are representative images of three replicates.
    Vegfr2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Post-radiation increase in VEGF enhances glioma cell motility in vitro"

    Article Title: Post-radiation increase in VEGF enhances glioma cell motility in vitro

    Journal: Radiation Oncology (London, England)

    doi: 10.1186/1748-717X-7-25

    The effects of irradiated conditioned medium (IR-CM) on activation of kinases in glioma cells . After 16 h incubation of GBM glioma cells with IR-CM ± monoclonal anti-human VEGF 165 antibody (R&D System), cells were harvested, processed to make lysates and the supernatant was collected. Equal quantities of protein were separated by SDS/PAGE and transferred to PVDF membrane. The membranes were blocked with 5% dried non-fat milk for 1 h at room temperature and incubated with the desired primary antibody diluted 1:1000 in blocking buffer overnight at 4 C. Membranes were probed with antibodies to phospho VEGFR2 Y996 , phospho VEGFR2 Y1056 , Src Y461 , phospho FAK Y861 and phospho FAK Y925 . Primary antibody incubation was followed by incubation with a horseradish peroxidase-conjugated secondary antibody in blocking buffer for 2 h at room temperature. Proteins were visualized with electrochemiluminescence detection reagents and detected by autoradiography. ( A ) IR-CM effect on phosphorylation of VEGFR2 in U251 glioma cells. ( B ) IR-CM with/without VEGF antibody (10 μg/ml) effect on the phosphorylation of Src and FAK in U251 glioma cells. Blots are representative images of three replicates.
    Figure Legend Snippet: The effects of irradiated conditioned medium (IR-CM) on activation of kinases in glioma cells . After 16 h incubation of GBM glioma cells with IR-CM ± monoclonal anti-human VEGF 165 antibody (R&D System), cells were harvested, processed to make lysates and the supernatant was collected. Equal quantities of protein were separated by SDS/PAGE and transferred to PVDF membrane. The membranes were blocked with 5% dried non-fat milk for 1 h at room temperature and incubated with the desired primary antibody diluted 1:1000 in blocking buffer overnight at 4 C. Membranes were probed with antibodies to phospho VEGFR2 Y996 , phospho VEGFR2 Y1056 , Src Y461 , phospho FAK Y861 and phospho FAK Y925 . Primary antibody incubation was followed by incubation with a horseradish peroxidase-conjugated secondary antibody in blocking buffer for 2 h at room temperature. Proteins were visualized with electrochemiluminescence detection reagents and detected by autoradiography. ( A ) IR-CM effect on phosphorylation of VEGFR2 in U251 glioma cells. ( B ) IR-CM with/without VEGF antibody (10 μg/ml) effect on the phosphorylation of Src and FAK in U251 glioma cells. Blots are representative images of three replicates.

    Techniques Used: Irradiation, Activation Assay, Incubation, SDS Page, Blocking Assay, Electrochemiluminescence, Autoradiography

    vegfr2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc vegfr2
    The effects of irradiated conditioned medium (IR-CM) on activation of kinases in glioma cells . After 16 h incubation of GBM glioma cells with IR-CM ± monoclonal anti-human VEGF 165 antibody (R&D System), cells were harvested, processed to make lysates and the supernatant was collected. Equal quantities of protein were separated by SDS/PAGE and transferred to PVDF membrane. The membranes were blocked with 5% dried non-fat milk for 1 h at room temperature and incubated with the desired primary antibody diluted 1:1000 in blocking buffer overnight at 4 C. Membranes were probed with antibodies to phospho <t>VEGFR2</t> Y996 , phospho VEGFR2 Y1056 , Src Y461 , phospho FAK Y861 and phospho FAK Y925 . Primary antibody incubation was followed by incubation with a horseradish peroxidase-conjugated secondary antibody in blocking buffer for 2 h at room temperature. Proteins were visualized with electrochemiluminescence detection reagents and detected by autoradiography. ( A ) IR-CM effect on phosphorylation of VEGFR2 in U251 glioma cells. ( B ) IR-CM with/without VEGF antibody (10 μg/ml) effect on the phosphorylation of Src and FAK in U251 glioma cells. Blots are representative images of three replicates.
    Vegfr2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Post-radiation increase in VEGF enhances glioma cell motility in vitro"

    Article Title: Post-radiation increase in VEGF enhances glioma cell motility in vitro

    Journal: Radiation Oncology (London, England)

    doi: 10.1186/1748-717X-7-25

    The effects of irradiated conditioned medium (IR-CM) on activation of kinases in glioma cells . After 16 h incubation of GBM glioma cells with IR-CM ± monoclonal anti-human VEGF 165 antibody (R&D System), cells were harvested, processed to make lysates and the supernatant was collected. Equal quantities of protein were separated by SDS/PAGE and transferred to PVDF membrane. The membranes were blocked with 5% dried non-fat milk for 1 h at room temperature and incubated with the desired primary antibody diluted 1:1000 in blocking buffer overnight at 4 C. Membranes were probed with antibodies to phospho VEGFR2 Y996 , phospho VEGFR2 Y1056 , Src Y461 , phospho FAK Y861 and phospho FAK Y925 . Primary antibody incubation was followed by incubation with a horseradish peroxidase-conjugated secondary antibody in blocking buffer for 2 h at room temperature. Proteins were visualized with electrochemiluminescence detection reagents and detected by autoradiography. ( A ) IR-CM effect on phosphorylation of VEGFR2 in U251 glioma cells. ( B ) IR-CM with/without VEGF antibody (10 μg/ml) effect on the phosphorylation of Src and FAK in U251 glioma cells. Blots are representative images of three replicates.
    Figure Legend Snippet: The effects of irradiated conditioned medium (IR-CM) on activation of kinases in glioma cells . After 16 h incubation of GBM glioma cells with IR-CM ± monoclonal anti-human VEGF 165 antibody (R&D System), cells were harvested, processed to make lysates and the supernatant was collected. Equal quantities of protein were separated by SDS/PAGE and transferred to PVDF membrane. The membranes were blocked with 5% dried non-fat milk for 1 h at room temperature and incubated with the desired primary antibody diluted 1:1000 in blocking buffer overnight at 4 C. Membranes were probed with antibodies to phospho VEGFR2 Y996 , phospho VEGFR2 Y1056 , Src Y461 , phospho FAK Y861 and phospho FAK Y925 . Primary antibody incubation was followed by incubation with a horseradish peroxidase-conjugated secondary antibody in blocking buffer for 2 h at room temperature. Proteins were visualized with electrochemiluminescence detection reagents and detected by autoradiography. ( A ) IR-CM effect on phosphorylation of VEGFR2 in U251 glioma cells. ( B ) IR-CM with/without VEGF antibody (10 μg/ml) effect on the phosphorylation of Src and FAK in U251 glioma cells. Blots are representative images of three replicates.

    Techniques Used: Irradiation, Activation Assay, Incubation, SDS Page, Blocking Assay, Electrochemiluminescence, Autoradiography

    anti vegfr2  (Cell Signaling Technology Inc)


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    anti src  (Cell Signaling Technology Inc)


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    vegfr2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc vegfr2
    A and B: MGO reduced <t>VEGFR2</t> protein levels in a time and dose dependent fashion. BAEC were incubated with MGO at indicated concentrations for up to 24 h. Then cells were subjected to Western blot respectively with a rabbit derived VEGFR2 antibody and a mouse derived β-actin antibody. All blots shown are representative of three independent experiments. *P<0.05 vs control (n = 3). C and D: MGO reduced endothelial cell angiogenesis in a dose dependent manner. Cells incubated with MGO (10–100 µM) were subjected to angiogenesis assessment by endothelial cell tube formation (upper panel in C) and migration (bottom panel in C). *P<0.05 vs control (n = 3).
    Vegfr2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Angiogenesis Impairment in Diabetes: Role of Methylglyoxal-Induced Receptor for Advanced Glycation Endproducts, Autophagy and Vascular Endothelial Growth Factor Receptor 2"

    Article Title: Angiogenesis Impairment in Diabetes: Role of Methylglyoxal-Induced Receptor for Advanced Glycation Endproducts, Autophagy and Vascular Endothelial Growth Factor Receptor 2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0046720

    A and B: MGO reduced VEGFR2 protein levels in a time and dose dependent fashion. BAEC were incubated with MGO at indicated concentrations for up to 24 h. Then cells were subjected to Western blot respectively with a rabbit derived VEGFR2 antibody and a mouse derived β-actin antibody. All blots shown are representative of three independent experiments. *P<0.05 vs control (n = 3). C and D: MGO reduced endothelial cell angiogenesis in a dose dependent manner. Cells incubated with MGO (10–100 µM) were subjected to angiogenesis assessment by endothelial cell tube formation (upper panel in C) and migration (bottom panel in C). *P<0.05 vs control (n = 3).
    Figure Legend Snippet: A and B: MGO reduced VEGFR2 protein levels in a time and dose dependent fashion. BAEC were incubated with MGO at indicated concentrations for up to 24 h. Then cells were subjected to Western blot respectively with a rabbit derived VEGFR2 antibody and a mouse derived β-actin antibody. All blots shown are representative of three independent experiments. *P<0.05 vs control (n = 3). C and D: MGO reduced endothelial cell angiogenesis in a dose dependent manner. Cells incubated with MGO (10–100 µM) were subjected to angiogenesis assessment by endothelial cell tube formation (upper panel in C) and migration (bottom panel in C). *P<0.05 vs control (n = 3).

    Techniques Used: Incubation, Western Blot, Derivative Assay, Migration

    A–B: MGO did not affect cell viability or apoptotic markers. BAEC were incubated with MGO at the indicated concentrations (for 24 h) or time course (up to 24 h) followed either by viability measurement (MTT assay) or Western blot staining for markers of apoptosis with respective antibodies against Bcl-2, Bax and Caspase 3. C: Knockdown of Glo1 by siRNA partly mimicked the MGO effect and enhanced MGO-induced VEGFR2 reduction. Transfection of control or Glo1 siRNA was performed based on protocols provided by Santa Cruz Biotechnology (Santa Cruz, CA). D: MGO increased levels of MGO-protein adduct which were further enhanced by siRNA knockdown of Glo1. E: Overexpression of Glo1 blocked MGO-induced VEGFR2 reduction. Infection of either control or Glo1 containing plasmid DNA was performed according to the protocol provided by OriGene (Rockville, MD). F: Overexpression of Glo1 blocked MGO-increased MGO-protein adducts. G: Knockdown of RAGE by siRNA prevented MGO-induced VEGFR2 reduction. Transfection of control or RAGE siRNA was performed based on protocols provided by Santa Cruz Biotechnology (Santa Cruz, CA). Western blot was performed with indicated antibodies. All blots shown are representative of three independent experiments. *P<0.05 vs control (n = 3). NS: not significant vs control (at 0 µM of MGO). Casp-3: Caspase-3; c-Casp-3: cleaved Caspase-3.
    Figure Legend Snippet: A–B: MGO did not affect cell viability or apoptotic markers. BAEC were incubated with MGO at the indicated concentrations (for 24 h) or time course (up to 24 h) followed either by viability measurement (MTT assay) or Western blot staining for markers of apoptosis with respective antibodies against Bcl-2, Bax and Caspase 3. C: Knockdown of Glo1 by siRNA partly mimicked the MGO effect and enhanced MGO-induced VEGFR2 reduction. Transfection of control or Glo1 siRNA was performed based on protocols provided by Santa Cruz Biotechnology (Santa Cruz, CA). D: MGO increased levels of MGO-protein adduct which were further enhanced by siRNA knockdown of Glo1. E: Overexpression of Glo1 blocked MGO-induced VEGFR2 reduction. Infection of either control or Glo1 containing plasmid DNA was performed according to the protocol provided by OriGene (Rockville, MD). F: Overexpression of Glo1 blocked MGO-increased MGO-protein adducts. G: Knockdown of RAGE by siRNA prevented MGO-induced VEGFR2 reduction. Transfection of control or RAGE siRNA was performed based on protocols provided by Santa Cruz Biotechnology (Santa Cruz, CA). Western blot was performed with indicated antibodies. All blots shown are representative of three independent experiments. *P<0.05 vs control (n = 3). NS: not significant vs control (at 0 µM of MGO). Casp-3: Caspase-3; c-Casp-3: cleaved Caspase-3.

    Techniques Used: Incubation, MTT Assay, Western Blot, Staining, Transfection, Over Expression, Infection, Plasmid Preparation

    A–E: Suppression of autophagy, but not proteasome and caspase, prevented VEGFR2 reduction induced by MGO. (A–C and E) One hour prior to MGO challenge (25 µM for 16 h), BAEC were pre-incubated respectively with chloroquine (CQ: 100 µM), pepstatin A (Pep A: 10 µM), bafilomycin A1 (Baf A1: 5 nM), MG132 (0.5 µM), epoxomicin (Epo: 0.5 µM), lactacystin (Lact: 1 µM), and z-VAD-fmk (z-VAD: 20 µM); (D) Before MGO treatment (as above), BAEC were transfected either with control siRNA or siRNA targeting Beclin-1, based on instructions from Santa Cruz Biotechnology (Santa Cruz, CA); all cell lysates were subjected to Western blot with indicated antibodies. All blots shown are representative of three independent experiments. *P<0.05 vs control (n = 3). F: Administration of autophagy inhibitor rescued MGO-impaired tube formation. One hour before MGO stimulation (25 µM), endothelial cells were incubated respectively with CQ (100 µM), Pep A (10 µM), Baf A1 (5 nM) and subjected to tube formation assay. G: Knockdown of Beclin 1 by siRNA prevented MGO-reduced tube formation. All images presented are representative of three independent experiments. *P<0.05 vs control (n = 3). NS: not significant vs control.
    Figure Legend Snippet: A–E: Suppression of autophagy, but not proteasome and caspase, prevented VEGFR2 reduction induced by MGO. (A–C and E) One hour prior to MGO challenge (25 µM for 16 h), BAEC were pre-incubated respectively with chloroquine (CQ: 100 µM), pepstatin A (Pep A: 10 µM), bafilomycin A1 (Baf A1: 5 nM), MG132 (0.5 µM), epoxomicin (Epo: 0.5 µM), lactacystin (Lact: 1 µM), and z-VAD-fmk (z-VAD: 20 µM); (D) Before MGO treatment (as above), BAEC were transfected either with control siRNA or siRNA targeting Beclin-1, based on instructions from Santa Cruz Biotechnology (Santa Cruz, CA); all cell lysates were subjected to Western blot with indicated antibodies. All blots shown are representative of three independent experiments. *P<0.05 vs control (n = 3). F: Administration of autophagy inhibitor rescued MGO-impaired tube formation. One hour before MGO stimulation (25 µM), endothelial cells were incubated respectively with CQ (100 µM), Pep A (10 µM), Baf A1 (5 nM) and subjected to tube formation assay. G: Knockdown of Beclin 1 by siRNA prevented MGO-reduced tube formation. All images presented are representative of three independent experiments. *P<0.05 vs control (n = 3). NS: not significant vs control.

    Techniques Used: Incubation, Transfection, Western Blot, Tube Formation Assay

    A: MGO increased autophagy markers Beclin 1 and LC3B. B: MGO induced LC3 punctae formation (autophagic flux) (Confocal microscopy imaging). C: MGO increased immunoprecipitation of VEGFR2 with LC3B, an autophagy marker and a component of the autophagosome. D: overexpression of Beclin 1 decreased VEGFR2 protein levels, and the reduction was accelerated when MGO was present. E: Rapamycin (1 µM for 1 h) decreased VEGFR2 protein levels. F: Rapamycin (1 µM) reduced endothelial tube formation. BAEC were incubated with MGO (25 µM) for 1 h (A–C) and 16 h (D). Cells were collected for Western blotting with a rabbit derived Beclin-1 or LC 3B antibody and a mouse derived β-actin antibody. All blots shown are representative of three independent experiments. *P<0.05 vs control (n = 3).
    Figure Legend Snippet: A: MGO increased autophagy markers Beclin 1 and LC3B. B: MGO induced LC3 punctae formation (autophagic flux) (Confocal microscopy imaging). C: MGO increased immunoprecipitation of VEGFR2 with LC3B, an autophagy marker and a component of the autophagosome. D: overexpression of Beclin 1 decreased VEGFR2 protein levels, and the reduction was accelerated when MGO was present. E: Rapamycin (1 µM for 1 h) decreased VEGFR2 protein levels. F: Rapamycin (1 µM) reduced endothelial tube formation. BAEC were incubated with MGO (25 µM) for 1 h (A–C) and 16 h (D). Cells were collected for Western blotting with a rabbit derived Beclin-1 or LC 3B antibody and a mouse derived β-actin antibody. All blots shown are representative of three independent experiments. *P<0.05 vs control (n = 3).

    Techniques Used: Confocal Microscopy, Imaging, Immunoprecipitation, Marker, Over Expression, Incubation, Western Blot, Derivative Assay

    − generation prevents reduction of VEGFR2 protein and endothelial cell angiogenesis by MGO. A–E: Suppression of ONOO − generation prevented reduction of VEGFR2 protein. BAEC were incubated respectively with vehicle (controls), mTempol (1 mM for 1 h), L-NAME (1 mM for 1 h), and UA (100 µM for 1 h), adenoviral infection of GFP (control), or SOD1 and SOD2, prior to MGO incubation (25 µM, 16 h). The treated cells were subjected to Western blot for VEGFR2 protein. All blots shown are representative of three independent experiments. *P<0.05 vs control (n = 3). F: Inhibition of ONOO − generation normalized the tube formation. Endothelial cells were incubated respectively with vehicle (controls), mTempol (1 mM for 1 h), L-NAME (1 mM for 1 h), and UA (100 µM for 1 h) prior to MGO challenge (25 µM). The treated cells were subjected to tube formation assay. The images shown are representative of three independent experiments. *P<0.05 vs control (n = 3). NS: not significant vs control.
    Figure Legend Snippet: − generation prevents reduction of VEGFR2 protein and endothelial cell angiogenesis by MGO. A–E: Suppression of ONOO − generation prevented reduction of VEGFR2 protein. BAEC were incubated respectively with vehicle (controls), mTempol (1 mM for 1 h), L-NAME (1 mM for 1 h), and UA (100 µM for 1 h), adenoviral infection of GFP (control), or SOD1 and SOD2, prior to MGO incubation (25 µM, 16 h). The treated cells were subjected to Western blot for VEGFR2 protein. All blots shown are representative of three independent experiments. *P<0.05 vs control (n = 3). F: Inhibition of ONOO − generation normalized the tube formation. Endothelial cells were incubated respectively with vehicle (controls), mTempol (1 mM for 1 h), L-NAME (1 mM for 1 h), and UA (100 µM for 1 h) prior to MGO challenge (25 µM). The treated cells were subjected to tube formation assay. The images shown are representative of three independent experiments. *P<0.05 vs control (n = 3). NS: not significant vs control.

    Techniques Used: Incubation, Infection, Western Blot, Inhibition, Tube Formation Assay

    A–B (low and high-power fields of the same slides): MGO reduced VEGFR2 immune-staining in cytosol or cytoplasm membrane, without affecting staining in the nucleus. BAEC were incubated with MGO (25 µM, 16 h) and subjected to cell immunofluorescent staining with a commercial immune-staining kit including ProLong® Gold and SlowFade® Gold Antifade, by using a rabbit derived VEGFR2 antibody or stained with DAPI, and a goat anti-rabbit IgG conjugate labeled with fluorescent dyes. All images shown are representative of three independent experiments. C: The quantification results of B. * P <0.05 vs control (n = 3). NS: not significant vs control.
    Figure Legend Snippet: A–B (low and high-power fields of the same slides): MGO reduced VEGFR2 immune-staining in cytosol or cytoplasm membrane, without affecting staining in the nucleus. BAEC were incubated with MGO (25 µM, 16 h) and subjected to cell immunofluorescent staining with a commercial immune-staining kit including ProLong® Gold and SlowFade® Gold Antifade, by using a rabbit derived VEGFR2 antibody or stained with DAPI, and a goat anti-rabbit IgG conjugate labeled with fluorescent dyes. All images shown are representative of three independent experiments. C: The quantification results of B. * P <0.05 vs control (n = 3). NS: not significant vs control.

    Techniques Used: Staining, Incubation, Derivative Assay, Labeling

    A: Aortic angiogenic response was reduced in MGO-challenged aortas of C57BL/6J mice, which could be restored by autophagy inhibitors. Aortic rings removed from normoglycemic C57BL/6J mice were incubated with MGO (25 µM) with or without the presence of autophagy inhibitors (Pep A: 10 µM, Baf A1: 5 µM) and subjected to aortic ring assay; * P <0.05 vs the vehicle treated (n = 5/group). B: Aortic endothelial outgrowth was impaired in db/db vs genetic control mice that could be rescued by autophagy inhibitors. Aortic rings prepared from the genetic control and db/db mice were treated with vehicle or autophagy inhibitors (Pep A: 10 µM, Baf A1: 5 µM) and followed by aortic ring assay. * P <0.05 vs the vehicle treated (n = 5/group). C–D: Aortic angiogenesis and VEGFR2 protein were reduced in diabetic Akita vs genetic control mice, which could be improved by mTempol administration in vivo . Aortas prepared from the genetic control and the treated Akita mice (mTempol: 0.1 mM in the drinking water, vehicle: normal drinking water, for 4 weeks) were subjected to aortic ring assay and aortic VEGFR2 protein detection by immune-precipitation (VEGFR2 enrichment)-combined Western blot. # indicates antibody recognizing different epitope of the VEGFR2 molecule. The Western blot of β-actin in aortic tissue homogenates with a mouse derived antibody is used as an Input for the loading control. * P <0.05 vs the vehicle treated (n = 5/group). E: the proposed mechanism of diabetic angiogenesis impairment: MGO, a glycolytic metabolite which is found to be increased in patients with diabetes, impairs endothelial angiogenesis through ONOO – dependent autophagy-mediated VEGFR2 degradation. NS: not significant vs control.
    Figure Legend Snippet: A: Aortic angiogenic response was reduced in MGO-challenged aortas of C57BL/6J mice, which could be restored by autophagy inhibitors. Aortic rings removed from normoglycemic C57BL/6J mice were incubated with MGO (25 µM) with or without the presence of autophagy inhibitors (Pep A: 10 µM, Baf A1: 5 µM) and subjected to aortic ring assay; * P <0.05 vs the vehicle treated (n = 5/group). B: Aortic endothelial outgrowth was impaired in db/db vs genetic control mice that could be rescued by autophagy inhibitors. Aortic rings prepared from the genetic control and db/db mice were treated with vehicle or autophagy inhibitors (Pep A: 10 µM, Baf A1: 5 µM) and followed by aortic ring assay. * P <0.05 vs the vehicle treated (n = 5/group). C–D: Aortic angiogenesis and VEGFR2 protein were reduced in diabetic Akita vs genetic control mice, which could be improved by mTempol administration in vivo . Aortas prepared from the genetic control and the treated Akita mice (mTempol: 0.1 mM in the drinking water, vehicle: normal drinking water, for 4 weeks) were subjected to aortic ring assay and aortic VEGFR2 protein detection by immune-precipitation (VEGFR2 enrichment)-combined Western blot. # indicates antibody recognizing different epitope of the VEGFR2 molecule. The Western blot of β-actin in aortic tissue homogenates with a mouse derived antibody is used as an Input for the loading control. * P <0.05 vs the vehicle treated (n = 5/group). E: the proposed mechanism of diabetic angiogenesis impairment: MGO, a glycolytic metabolite which is found to be increased in patients with diabetes, impairs endothelial angiogenesis through ONOO – dependent autophagy-mediated VEGFR2 degradation. NS: not significant vs control.

    Techniques Used: Incubation, Aortic Ring Assay, In Vivo, Western Blot, Derivative Assay

    anti fak tyr  (Cell Signaling Technology Inc)


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    Anti Fak Tyr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    vegfr2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc vegfr2
    (A) EC, EC IRES GFP and EC <t>VEGFR2</t> IRES GFP were cultured in growing media and VEGFR2 overexpression was analysed by immunofluorescence. Cells were stained with a rabbit anti-human VEGFR2 antibody (Alexa 594). Results shown are representative z-projections of at least three independent experiments. Scale bar: 20 µm. Right panel shows mean fluorescence intensity of VEGFR2 in the cell nucleus. * p <0.0001. (B and C) FRAP analysis was performed in EC VEGFR2-GFP and mutants EC VEGFR2(Y1054F/Y1059F)-GFP, EC VEGFR2(Y996F)-GFP and EC VEGFR2(Y951F)-GFP. Fluorescence signal of the entire nucleus was photobleached with a single 488-nm high intensity laser pulse and subsequent fluorescence recovery was recorded for 280 s. (B) Selected images of VEGFR2-GFP protein in EC VEGFR2-GFP (upper panel) and EC VEGFR2(Y951F)-GFP (lower panel) before bleaching (steady-state) at the indicated intervals post-bleaching (from 5 to 100 s). White circles indicate the bleached region. (C) Fluorescence intensity in the bleached region was measured every 5 s for 280 s and normalized for the initial intensity. Data show results obtained in three independent experiments, with at least ten different cells analysed in each case. Error bars represent standard deviation (SD).
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    1) Product Images from "VEGFR2 Translocates to the Nucleus to Regulate Its Own Transcription"

    Article Title: VEGFR2 Translocates to the Nucleus to Regulate Its Own Transcription

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025668

    (A) EC, EC IRES GFP and EC VEGFR2 IRES GFP were cultured in growing media and VEGFR2 overexpression was analysed by immunofluorescence. Cells were stained with a rabbit anti-human VEGFR2 antibody (Alexa 594). Results shown are representative z-projections of at least three independent experiments. Scale bar: 20 µm. Right panel shows mean fluorescence intensity of VEGFR2 in the cell nucleus. * p <0.0001. (B and C) FRAP analysis was performed in EC VEGFR2-GFP and mutants EC VEGFR2(Y1054F/Y1059F)-GFP, EC VEGFR2(Y996F)-GFP and EC VEGFR2(Y951F)-GFP. Fluorescence signal of the entire nucleus was photobleached with a single 488-nm high intensity laser pulse and subsequent fluorescence recovery was recorded for 280 s. (B) Selected images of VEGFR2-GFP protein in EC VEGFR2-GFP (upper panel) and EC VEGFR2(Y951F)-GFP (lower panel) before bleaching (steady-state) at the indicated intervals post-bleaching (from 5 to 100 s). White circles indicate the bleached region. (C) Fluorescence intensity in the bleached region was measured every 5 s for 280 s and normalized for the initial intensity. Data show results obtained in three independent experiments, with at least ten different cells analysed in each case. Error bars represent standard deviation (SD).
    Figure Legend Snippet: (A) EC, EC IRES GFP and EC VEGFR2 IRES GFP were cultured in growing media and VEGFR2 overexpression was analysed by immunofluorescence. Cells were stained with a rabbit anti-human VEGFR2 antibody (Alexa 594). Results shown are representative z-projections of at least three independent experiments. Scale bar: 20 µm. Right panel shows mean fluorescence intensity of VEGFR2 in the cell nucleus. * p <0.0001. (B and C) FRAP analysis was performed in EC VEGFR2-GFP and mutants EC VEGFR2(Y1054F/Y1059F)-GFP, EC VEGFR2(Y996F)-GFP and EC VEGFR2(Y951F)-GFP. Fluorescence signal of the entire nucleus was photobleached with a single 488-nm high intensity laser pulse and subsequent fluorescence recovery was recorded for 280 s. (B) Selected images of VEGFR2-GFP protein in EC VEGFR2-GFP (upper panel) and EC VEGFR2(Y951F)-GFP (lower panel) before bleaching (steady-state) at the indicated intervals post-bleaching (from 5 to 100 s). White circles indicate the bleached region. (C) Fluorescence intensity in the bleached region was measured every 5 s for 280 s and normalized for the initial intensity. Data show results obtained in three independent experiments, with at least ten different cells analysed in each case. Error bars represent standard deviation (SD).

    Techniques Used: Cell Culture, Over Expression, Immunofluorescence, Staining, Fluorescence, Standard Deviation

    (A) Cytoplasmic (C) and nuclear extracts (N) from EC IRES GFP and EC VEGFR2 IRES GFP were analysed by Immunoblot with antibodies against VEGFR2, Cyclin A, Sp1, p65, YY1. P-IkB and Lamin B were used as cytoplasmic and nuclear controls, respectively. (B) Nuclear extracts from EC IRES GFP (lanes 2,7) and EC VEGFR2 IRES GFP (lanes 3,8) were incubated with NFkB (left panel, lanes 2 and 3) or YY1 (right panel, lanes 7 and 8) radiolabeled probes. Four NFkB (C1–C4) or five YY1 complexes (C1–C5) are indicated with black arrows. Specific anti-p65 (lane 4) or anti-YY1 (lane 9) were introduced in the binding reaction to analyse the appearance of a supershift complex (as indicated in both panels) in EC VEGFR2 IRES GFP cells. Using the same cells, a competitive assay using 100x excess of cold probe of NFkB (lane 5) or YY1 (lane10) was performed. Control lanes 1 and 6 contain only the radiolabeled probes. (C) EC were cultured in growing media, treated or not with 6.12 Ab for 1 h and incubated with 5-FU for 15 min. (D) EC were cultured in growing media and transfected with scrambled siRNA or VEGFR2 siRNA. EC were incubated with 5-FU for 15 min. (C and D) 24 post-transfection. Cells were fixed and sequentially labeled on the same slide with a rabbit anti-human VEGFR2 (red fluorescence) and a mouse anti-human BrdU antibody (green fluorescence) and analysed by confocal microscopy. Results shown are representative z-projections of three independent experiments. Scale bar: 20 µm.
    Figure Legend Snippet: (A) Cytoplasmic (C) and nuclear extracts (N) from EC IRES GFP and EC VEGFR2 IRES GFP were analysed by Immunoblot with antibodies against VEGFR2, Cyclin A, Sp1, p65, YY1. P-IkB and Lamin B were used as cytoplasmic and nuclear controls, respectively. (B) Nuclear extracts from EC IRES GFP (lanes 2,7) and EC VEGFR2 IRES GFP (lanes 3,8) were incubated with NFkB (left panel, lanes 2 and 3) or YY1 (right panel, lanes 7 and 8) radiolabeled probes. Four NFkB (C1–C4) or five YY1 complexes (C1–C5) are indicated with black arrows. Specific anti-p65 (lane 4) or anti-YY1 (lane 9) were introduced in the binding reaction to analyse the appearance of a supershift complex (as indicated in both panels) in EC VEGFR2 IRES GFP cells. Using the same cells, a competitive assay using 100x excess of cold probe of NFkB (lane 5) or YY1 (lane10) was performed. Control lanes 1 and 6 contain only the radiolabeled probes. (C) EC were cultured in growing media, treated or not with 6.12 Ab for 1 h and incubated with 5-FU for 15 min. (D) EC were cultured in growing media and transfected with scrambled siRNA or VEGFR2 siRNA. EC were incubated with 5-FU for 15 min. (C and D) 24 post-transfection. Cells were fixed and sequentially labeled on the same slide with a rabbit anti-human VEGFR2 (red fluorescence) and a mouse anti-human BrdU antibody (green fluorescence) and analysed by confocal microscopy. Results shown are representative z-projections of three independent experiments. Scale bar: 20 µm.

    Techniques Used: Western Blot, Incubation, Binding Assay, Cell Culture, Transfection, Labeling, Fluorescence, Confocal Microscopy

    (A) Immunoprecipitation (IP) of 1 mg EC nuclear extract with anti-human VEGFR2 was performed and resolved in 8% SDS-PAGE, following silver staining (lane 2). VEGFR2 antibody plus beads (without N) were used as negative control for immunoprecipitation (lane 1). The protein marker is shown as molecular weight (MW) in thousands. (B) Representation of the mass spectrometry analysis of the nuclear VEGFR2 IP, showing the categories for the different biological functions of the identified proteins (p<0.05). (C) Immunoprecipitation (IP) of EC nuclear extracts (N) was conducted with the VEGFR2 (lane 2), Sp1 (lane 4) and rabbit IgG (rIgG-lane 5) antibodies followed by VEGFR2 (upper panel) or Sp1 (lower panel) immunoblotting. VEGFR2 (lane 1) or Sp1 (lane 3) antibodies plus beads (without N) were used as negative controls for immunoprecipitation. Non-immunoprecipitated nuclear cell extract (lane 6) was also included in the experiment. (D) Pull-down assay: Sp1 protein fused to a HA tag was incubated with GST alone (lanes 1 and 2) or VEGFR2 (789-1356)-GST (lanes 3 and 4). GST-unbound (UB) (lanes 1 and 3) and bound (B) fractions (lanes 2 and 4) were loaded and analyzed with GST (upper panel) and Sp1 (lower panel) antibodies.
    Figure Legend Snippet: (A) Immunoprecipitation (IP) of 1 mg EC nuclear extract with anti-human VEGFR2 was performed and resolved in 8% SDS-PAGE, following silver staining (lane 2). VEGFR2 antibody plus beads (without N) were used as negative control for immunoprecipitation (lane 1). The protein marker is shown as molecular weight (MW) in thousands. (B) Representation of the mass spectrometry analysis of the nuclear VEGFR2 IP, showing the categories for the different biological functions of the identified proteins (p<0.05). (C) Immunoprecipitation (IP) of EC nuclear extracts (N) was conducted with the VEGFR2 (lane 2), Sp1 (lane 4) and rabbit IgG (rIgG-lane 5) antibodies followed by VEGFR2 (upper panel) or Sp1 (lower panel) immunoblotting. VEGFR2 (lane 1) or Sp1 (lane 3) antibodies plus beads (without N) were used as negative controls for immunoprecipitation. Non-immunoprecipitated nuclear cell extract (lane 6) was also included in the experiment. (D) Pull-down assay: Sp1 protein fused to a HA tag was incubated with GST alone (lanes 1 and 2) or VEGFR2 (789-1356)-GST (lanes 3 and 4). GST-unbound (UB) (lanes 1 and 3) and bound (B) fractions (lanes 2 and 4) were loaded and analyzed with GST (upper panel) and Sp1 (lower panel) antibodies.

    Techniques Used: Immunoprecipitation, SDS Page, Silver Staining, Negative Control, Marker, Molecular Weight, Mass Spectrometry, Western Blot, Pull Down Assay, Incubation

    (A) Sequence of the human VEGFR2 proximal promoter (retrieved from Ensemble database accession number: ENSG00000128052) and outline of putative Sp1 binding sites. The transcription start site is indicated in gray. (B) ChIP assays of the VEGFR2 proximal promoter were performed using EC cultured in growing media. Antibodies against VEGFR2 and Sp1 were used. Normal rabbit/mouse IgG were used as control. Also, an antibody for RNA Pol II was used to test the promoter activity. All values are relative to control IgG background and normalized to an intergenic region. Data are mean ± s.e.m. of triplicates and represents three independent experiments. (C) ChIP assays of the VEGFR2 proximal promoter were performed in EC 24 h post-transfection of scrambled siRNA or VEGFR2 siRNA or Sp1 siRNA. Antibodies against VEGFR2 and Sp1 were used. Normal rabbit IgG was used as control. Values are relative to control IgG background and normalized to an intergenic region. Data are mean ± s.e.m. of triplicates and represents three independent experiments. (D) EMSA analysis of the VEGFR2 promoter with EC nuclear extracts (lane 2) or VEGFR2 immunodepleted (ID VEGFR2) extract (lane 3) were conducted. Four complexes (C1-C4) are indicated with black arrows. A competitive assay using 100x excess of cold probe of VEGFR2 promoter was conducted (lane 4) using EC nuclear extract. Control lane 1 contains only the radiolabeled probe.
    Figure Legend Snippet: (A) Sequence of the human VEGFR2 proximal promoter (retrieved from Ensemble database accession number: ENSG00000128052) and outline of putative Sp1 binding sites. The transcription start site is indicated in gray. (B) ChIP assays of the VEGFR2 proximal promoter were performed using EC cultured in growing media. Antibodies against VEGFR2 and Sp1 were used. Normal rabbit/mouse IgG were used as control. Also, an antibody for RNA Pol II was used to test the promoter activity. All values are relative to control IgG background and normalized to an intergenic region. Data are mean ± s.e.m. of triplicates and represents three independent experiments. (C) ChIP assays of the VEGFR2 proximal promoter were performed in EC 24 h post-transfection of scrambled siRNA or VEGFR2 siRNA or Sp1 siRNA. Antibodies against VEGFR2 and Sp1 were used. Normal rabbit IgG was used as control. Values are relative to control IgG background and normalized to an intergenic region. Data are mean ± s.e.m. of triplicates and represents three independent experiments. (D) EMSA analysis of the VEGFR2 promoter with EC nuclear extracts (lane 2) or VEGFR2 immunodepleted (ID VEGFR2) extract (lane 3) were conducted. Four complexes (C1-C4) are indicated with black arrows. A competitive assay using 100x excess of cold probe of VEGFR2 promoter was conducted (lane 4) using EC nuclear extract. Control lane 1 contains only the radiolabeled probe.

    Techniques Used: Sequencing, Binding Assay, Cell Culture, Activity Assay, Transfection

    (A) NIH 3T3 GFP and NIH 3T3 VEGFR2-GFP were transfected with pGL3 control or pGL3 VEGFR2 (-300/+1) or pGL3 VEGFR2 (-116/+1). The β-gal plasmid was co-transfected as a control. Promoter activities were measured with luciferase activity normalized to β-gal. The results are expressed as the relative luciferase activities. Data are mean ± s.e.m. of relative luciferase activities from four independent experiments, each performed in triplicate. * p = 0.007; ** p = 0.001). (B) NIH 3T3 VEGFR2-GFP were transfected with scrambled siRNA, VEGFR2 siRNA or Sp1 siRNA. At 48 h post-transfection the relative luciferase activity of pGL3 control or pGL3 VEGFR2 (-300/+1) was measured. Data are mean ± s.e.m. of relative luciferase activities from three independent experiments, each performed in triplicate, (* p = 0.003).
    Figure Legend Snippet: (A) NIH 3T3 GFP and NIH 3T3 VEGFR2-GFP were transfected with pGL3 control or pGL3 VEGFR2 (-300/+1) or pGL3 VEGFR2 (-116/+1). The β-gal plasmid was co-transfected as a control. Promoter activities were measured with luciferase activity normalized to β-gal. The results are expressed as the relative luciferase activities. Data are mean ± s.e.m. of relative luciferase activities from four independent experiments, each performed in triplicate. * p = 0.007; ** p = 0.001). (B) NIH 3T3 VEGFR2-GFP were transfected with scrambled siRNA, VEGFR2 siRNA or Sp1 siRNA. At 48 h post-transfection the relative luciferase activity of pGL3 control or pGL3 VEGFR2 (-300/+1) was measured. Data are mean ± s.e.m. of relative luciferase activities from three independent experiments, each performed in triplicate, (* p = 0.003).

    Techniques Used: Transfection, Plasmid Preparation, Luciferase, Activity Assay

    ChIP assays of the VEGFR2 proximal promoter were performed using (A) EC cultured in basal medium for 48 h, and stimulated or not with VEGF (20 ng/ml) for 30 min. Antibodies against VEGFR2 and Sp1 were used. Normal rabbit/mouse IgG were used as control. Also, an antibody for RNA Pol II was used to test the promoter activity. Values are relative to control IgG background and normalized to an intergenic region. Data are mean ± s.e.m. of triplicates and represents three independent experiments. (B) EC cultured in growing media were treated or not with 0.5 mg/ml Bevacizumab (left panel) or 0.1 µM Sunitinib (right panel) for 16 h. In the Sunitinib experiments, DMSO was used as vehicle. ChIP values are relative to control IgG and normalized to an intergenic region. Data are mean ± s.e.m. of triplicates and represents three independent experiments.
    Figure Legend Snippet: ChIP assays of the VEGFR2 proximal promoter were performed using (A) EC cultured in basal medium for 48 h, and stimulated or not with VEGF (20 ng/ml) for 30 min. Antibodies against VEGFR2 and Sp1 were used. Normal rabbit/mouse IgG were used as control. Also, an antibody for RNA Pol II was used to test the promoter activity. Values are relative to control IgG background and normalized to an intergenic region. Data are mean ± s.e.m. of triplicates and represents three independent experiments. (B) EC cultured in growing media were treated or not with 0.5 mg/ml Bevacizumab (left panel) or 0.1 µM Sunitinib (right panel) for 16 h. In the Sunitinib experiments, DMSO was used as vehicle. ChIP values are relative to control IgG and normalized to an intergenic region. Data are mean ± s.e.m. of triplicates and represents three independent experiments.

    Techniques Used: Cell Culture, Activity Assay

    anti akt antibodies  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti akt antibodies
    Growth factor-starved primary HCECs cells were either left unchallenged (CT) or challenged with purified flagellin (250 ng/ml) from indicated bacterial strains; PF(wild type PAK), LF (L94A), QF (Q83A), and then lysed 60 min post-stimulation. Equal amounts of proteins of 30–45 µg were subjected to Western blot using antibodies against phospho- (p) <t>proteins</t> <t>IκB-α,</t> P38, EGFR Y 1173 , ERK or <t>AKT.</t> After stripping off the immunoreactivities, the membranes were reprobed with antibodies against, corresponding proteins for proper protein loading. The Figure is a representative of three independent experiments.
    Anti Akt Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Flagellin-Induced Corneal Antimicrobial Peptide Production and Wound Repair Involve a Novel NF-κB–Independent and EGFR-Dependent Pathway"

    Article Title: Flagellin-Induced Corneal Antimicrobial Peptide Production and Wound Repair Involve a Novel NF-κB–Independent and EGFR-Dependent Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0009351

    Growth factor-starved primary HCECs cells were either left unchallenged (CT) or challenged with purified flagellin (250 ng/ml) from indicated bacterial strains; PF(wild type PAK), LF (L94A), QF (Q83A), and then lysed 60 min post-stimulation. Equal amounts of proteins of 30–45 µg were subjected to Western blot using antibodies against phospho- (p) proteins IκB-α, P38, EGFR Y 1173 , ERK or AKT. After stripping off the immunoreactivities, the membranes were reprobed with antibodies against, corresponding proteins for proper protein loading. The Figure is a representative of three independent experiments.
    Figure Legend Snippet: Growth factor-starved primary HCECs cells were either left unchallenged (CT) or challenged with purified flagellin (250 ng/ml) from indicated bacterial strains; PF(wild type PAK), LF (L94A), QF (Q83A), and then lysed 60 min post-stimulation. Equal amounts of proteins of 30–45 µg were subjected to Western blot using antibodies against phospho- (p) proteins IκB-α, P38, EGFR Y 1173 , ERK or AKT. After stripping off the immunoreactivities, the membranes were reprobed with antibodies against, corresponding proteins for proper protein loading. The Figure is a representative of three independent experiments.

    Techniques Used: Purification, Western Blot, Stripping Membranes

    Growth factor-starved HUCL cells were pretreated with AG1478 (AG, 1 µM), CRM197 (CRM, 10 ng/µl), or TGF-α neutralizing antibody (α-TGF-α 5 µg/ml) for 1 h and then challenged with purified wild type PAK flagellin (PF). One hour post flagellin challenge, cells were lysed and equal amounts of proteins of 30–45 µg were subjected to Western blot using antibodies against phospho- (p) proteins JNK, EGFR at Y 1173 , ERK or AKT. After stripping off the immunoreactivities, the membranes were reprobed with antibodies against proteins for proper loading. The Figure is a representative of two independent experiments.
    Figure Legend Snippet: Growth factor-starved HUCL cells were pretreated with AG1478 (AG, 1 µM), CRM197 (CRM, 10 ng/µl), or TGF-α neutralizing antibody (α-TGF-α 5 µg/ml) for 1 h and then challenged with purified wild type PAK flagellin (PF). One hour post flagellin challenge, cells were lysed and equal amounts of proteins of 30–45 µg were subjected to Western blot using antibodies against phospho- (p) proteins JNK, EGFR at Y 1173 , ERK or AKT. After stripping off the immunoreactivities, the membranes were reprobed with antibodies against proteins for proper loading. The Figure is a representative of two independent experiments.

    Techniques Used: Purification, Western Blot, Stripping Membranes

    HUCL cells were either pretreated with 50 ng/ml PAK (PF) or L94A (LF) flagellin or left untreated (C) for 24 h and then challenged with (PAK) or without (Control) live PAK for 1 h. Total cell lysates were blotted with antibodies specific for phospho-ERK, AKT, IκB-α, and P38, followed by reprobing with antibodies against corresponding proteins. The data shown are representative of 2 independent experiments.
    Figure Legend Snippet: HUCL cells were either pretreated with 50 ng/ml PAK (PF) or L94A (LF) flagellin or left untreated (C) for 24 h and then challenged with (PAK) or without (Control) live PAK for 1 h. Total cell lysates were blotted with antibodies specific for phospho-ERK, AKT, IκB-α, and P38, followed by reprobing with antibodies against corresponding proteins. The data shown are representative of 2 independent experiments.

    Techniques Used:

    Epithelial cells in healing porcine corneas treated with wild type (PF), L94A (LF) flagellin, or medium alone (CT), were collected 48 hrs post wounding, lysed in RIPA buffer and sonicated. Equal amounts of cell lysates from 2 of 5 corneas (randomly picked) from each group were immunoblotted with antibodies against phospho-EGFR, AKT or ERK1/2. The immunoreactivities were stripped off and the same membranes were reprobed with antibodies against cellular EGFR, AKT or ERK2, respectively, for proper protein loading.
    Figure Legend Snippet: Epithelial cells in healing porcine corneas treated with wild type (PF), L94A (LF) flagellin, or medium alone (CT), were collected 48 hrs post wounding, lysed in RIPA buffer and sonicated. Equal amounts of cell lysates from 2 of 5 corneas (randomly picked) from each group were immunoblotted with antibodies against phospho-EGFR, AKT or ERK1/2. The immunoreactivities were stripped off and the same membranes were reprobed with antibodies against cellular EGFR, AKT or ERK2, respectively, for proper protein loading.

    Techniques Used: Sonication

    p85  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p85
    Effect of BAY on the activation of upstream signaling for NF- κ B translocation. (a to c) RAW264.7 cells (a left panel, b, and c) or peritoneal macrophages (a right panel) (5 × 10 6 cells/mL) were incubated with BAY (15 μ M) in the presence or absence of LPS (1 μ g/mL) for indicated times. After preparing whole lysates, levels of phospho (p)- or total proteins of I κ B α , IKK α / β , Akt, <t>p85/PI3K,</t> Syk, and Src were identified by immunoprecipitation. Relative intensity (a right panel) was calculated by densitometric scanning.
    P85, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "BAY 11-7082 Is a Broad-Spectrum Inhibitor with Anti-Inflammatory Activity against Multiple Targets"

    Article Title: BAY 11-7082 Is a Broad-Spectrum Inhibitor with Anti-Inflammatory Activity against Multiple Targets

    Journal: Mediators of Inflammation

    doi: 10.1155/2012/416036

    Effect of BAY on the activation of upstream signaling for NF- κ B translocation. (a to c) RAW264.7 cells (a left panel, b, and c) or peritoneal macrophages (a right panel) (5 × 10 6 cells/mL) were incubated with BAY (15 μ M) in the presence or absence of LPS (1 μ g/mL) for indicated times. After preparing whole lysates, levels of phospho (p)- or total proteins of I κ B α , IKK α / β , Akt, p85/PI3K, Syk, and Src were identified by immunoprecipitation. Relative intensity (a right panel) was calculated by densitometric scanning.
    Figure Legend Snippet: Effect of BAY on the activation of upstream signaling for NF- κ B translocation. (a to c) RAW264.7 cells (a left panel, b, and c) or peritoneal macrophages (a right panel) (5 × 10 6 cells/mL) were incubated with BAY (15 μ M) in the presence or absence of LPS (1 μ g/mL) for indicated times. After preparing whole lysates, levels of phospho (p)- or total proteins of I κ B α , IKK α / β , Akt, p85/PI3K, Syk, and Src were identified by immunoprecipitation. Relative intensity (a right panel) was calculated by densitometric scanning.

    Techniques Used: Activation Assay, Translocation Assay, Incubation, Immunoprecipitation

    anti vegfr2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti vegfr2
    Anti Vegfr2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti akt  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti akt
    ( A ) L6 myotubes were incubated with serum-free media containing 2% (w/v) BSA (vehicle) ± 700 μM (A) oleic acid (OA) or (B) linoleic acid (LOA) for 16h prior to stimulation with insulin at concentrations indicated (10min). ( C, D ) L6 myotubes were incubated as in (A) prior to stimulation with insulin (20 nM, 10min). ( E ) Human myotubes were incubated with 200 μM OA or LOA for 24h prior to stimulation with insulin (20 nM, 10min). Cell lysates from A-E were subsequently immunoblotted using antibodies against the proteins indicated with quantification of <t>phospho-Akt</t> (Ser473 and Thr308) <t>and</t> <t>phospho-ERK1/2</t> shown in panel A,B and D expressed as mean ± SEM of at least three experiments. The immunoblots shown in panel E are representative of two separate experiments. The asterisks signify a significant change (p<0.05) between the indicated bars (A,B) or the insulin-treatment alone (D).
    Anti Akt, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Enhanced Insulin Sensitivity Associated with Provision of Mono and Polyunsaturated Fatty Acids in Skeletal Muscle Cells Involves Counter Modulation of PP2A"

    Article Title: Enhanced Insulin Sensitivity Associated with Provision of Mono and Polyunsaturated Fatty Acids in Skeletal Muscle Cells Involves Counter Modulation of PP2A

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0092255

    ( A ) L6 myotubes were incubated with serum-free media containing 2% (w/v) BSA (vehicle) ± 700 μM (A) oleic acid (OA) or (B) linoleic acid (LOA) for 16h prior to stimulation with insulin at concentrations indicated (10min). ( C, D ) L6 myotubes were incubated as in (A) prior to stimulation with insulin (20 nM, 10min). ( E ) Human myotubes were incubated with 200 μM OA or LOA for 24h prior to stimulation with insulin (20 nM, 10min). Cell lysates from A-E were subsequently immunoblotted using antibodies against the proteins indicated with quantification of phospho-Akt (Ser473 and Thr308) and phospho-ERK1/2 shown in panel A,B and D expressed as mean ± SEM of at least three experiments. The immunoblots shown in panel E are representative of two separate experiments. The asterisks signify a significant change (p<0.05) between the indicated bars (A,B) or the insulin-treatment alone (D).
    Figure Legend Snippet: ( A ) L6 myotubes were incubated with serum-free media containing 2% (w/v) BSA (vehicle) ± 700 μM (A) oleic acid (OA) or (B) linoleic acid (LOA) for 16h prior to stimulation with insulin at concentrations indicated (10min). ( C, D ) L6 myotubes were incubated as in (A) prior to stimulation with insulin (20 nM, 10min). ( E ) Human myotubes were incubated with 200 μM OA or LOA for 24h prior to stimulation with insulin (20 nM, 10min). Cell lysates from A-E were subsequently immunoblotted using antibodies against the proteins indicated with quantification of phospho-Akt (Ser473 and Thr308) and phospho-ERK1/2 shown in panel A,B and D expressed as mean ± SEM of at least three experiments. The immunoblots shown in panel E are representative of two separate experiments. The asterisks signify a significant change (p<0.05) between the indicated bars (A,B) or the insulin-treatment alone (D).

    Techniques Used: Incubation, Western Blot

    The experimental approach used to test the effect of OA and LOA provision on Akt and ERK1/2 phosphorylation is depicted in (A). L6 myotubes were incubated with serum-free media containing 2% (w/v) BSA (vehicle) ± 700 μM (B and C) oleic acid (OA) or (D and E) linoleic acid (LOA) for 16h prior to stimulation with insulin (20 nM, 10min). Cells were washed to remove insulin and subsequently incubated with 100 nM wortmannin (WM) and lysed at times indicated. Immunoblots and relevant quantification of phospho-Akt and phospho-ERK1/2 in L6 myotubes incubated with OA (B,C) or LOA (D,E). Data in the bar graphs is presented as mean ± SEM (n = 3) with asterisks indicating a significant change (p<0.05) between the respective filled (fatty acid treatment) and unfilled (control) bar at each time point.
    Figure Legend Snippet: The experimental approach used to test the effect of OA and LOA provision on Akt and ERK1/2 phosphorylation is depicted in (A). L6 myotubes were incubated with serum-free media containing 2% (w/v) BSA (vehicle) ± 700 μM (B and C) oleic acid (OA) or (D and E) linoleic acid (LOA) for 16h prior to stimulation with insulin (20 nM, 10min). Cells were washed to remove insulin and subsequently incubated with 100 nM wortmannin (WM) and lysed at times indicated. Immunoblots and relevant quantification of phospho-Akt and phospho-ERK1/2 in L6 myotubes incubated with OA (B,C) or LOA (D,E). Data in the bar graphs is presented as mean ± SEM (n = 3) with asterisks indicating a significant change (p<0.05) between the respective filled (fatty acid treatment) and unfilled (control) bar at each time point.

    Techniques Used: Incubation, Western Blot

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    Cell Signaling Technology Inc vegfr2
    The effects of irradiated conditioned medium (IR-CM) on activation of kinases in glioma cells . After 16 h incubation of GBM glioma cells with IR-CM ± monoclonal anti-human VEGF 165 antibody (R&D System), cells were harvested, processed to make lysates and the supernatant was collected. Equal quantities of protein were separated by SDS/PAGE and transferred to PVDF membrane. The membranes were blocked with 5% dried non-fat milk for 1 h at room temperature and incubated with the desired primary antibody diluted 1:1000 in blocking buffer overnight at 4 C. Membranes were probed with antibodies to phospho <t>VEGFR2</t> Y996 , phospho VEGFR2 Y1056 , Src Y461 , phospho FAK Y861 and phospho FAK Y925 . Primary antibody incubation was followed by incubation with a horseradish peroxidase-conjugated secondary antibody in blocking buffer for 2 h at room temperature. Proteins were visualized with electrochemiluminescence detection reagents and detected by autoradiography. ( A ) IR-CM effect on phosphorylation of VEGFR2 in U251 glioma cells. ( B ) IR-CM with/without VEGF antibody (10 μg/ml) effect on the phosphorylation of Src and FAK in U251 glioma cells. Blots are representative images of three replicates.
    Vegfr2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti vegfr2
    The effects of irradiated conditioned medium (IR-CM) on activation of kinases in glioma cells . After 16 h incubation of GBM glioma cells with IR-CM ± monoclonal anti-human VEGF 165 antibody (R&D System), cells were harvested, processed to make lysates and the supernatant was collected. Equal quantities of protein were separated by SDS/PAGE and transferred to PVDF membrane. The membranes were blocked with 5% dried non-fat milk for 1 h at room temperature and incubated with the desired primary antibody diluted 1:1000 in blocking buffer overnight at 4 C. Membranes were probed with antibodies to phospho <t>VEGFR2</t> Y996 , phospho VEGFR2 Y1056 , Src Y461 , phospho FAK Y861 and phospho FAK Y925 . Primary antibody incubation was followed by incubation with a horseradish peroxidase-conjugated secondary antibody in blocking buffer for 2 h at room temperature. Proteins were visualized with electrochemiluminescence detection reagents and detected by autoradiography. ( A ) IR-CM effect on phosphorylation of VEGFR2 in U251 glioma cells. ( B ) IR-CM with/without VEGF antibody (10 μg/ml) effect on the phosphorylation of Src and FAK in U251 glioma cells. Blots are representative images of three replicates.
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    Cell Signaling Technology Inc anti src
    The effects of irradiated conditioned medium (IR-CM) on activation of kinases in glioma cells . After 16 h incubation of GBM glioma cells with IR-CM ± monoclonal anti-human VEGF 165 antibody (R&D System), cells were harvested, processed to make lysates and the supernatant was collected. Equal quantities of protein were separated by SDS/PAGE and transferred to PVDF membrane. The membranes were blocked with 5% dried non-fat milk for 1 h at room temperature and incubated with the desired primary antibody diluted 1:1000 in blocking buffer overnight at 4 C. Membranes were probed with antibodies to phospho <t>VEGFR2</t> Y996 , phospho VEGFR2 Y1056 , Src Y461 , phospho FAK Y861 and phospho FAK Y925 . Primary antibody incubation was followed by incubation with a horseradish peroxidase-conjugated secondary antibody in blocking buffer for 2 h at room temperature. Proteins were visualized with electrochemiluminescence detection reagents and detected by autoradiography. ( A ) IR-CM effect on phosphorylation of VEGFR2 in U251 glioma cells. ( B ) IR-CM with/without VEGF antibody (10 μg/ml) effect on the phosphorylation of Src and FAK in U251 glioma cells. Blots are representative images of three replicates.
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    Cell Signaling Technology Inc anti fak tyr
    The effects of irradiated conditioned medium (IR-CM) on activation of kinases in glioma cells . After 16 h incubation of GBM glioma cells with IR-CM ± monoclonal anti-human VEGF 165 antibody (R&D System), cells were harvested, processed to make lysates and the supernatant was collected. Equal quantities of protein were separated by SDS/PAGE and transferred to PVDF membrane. The membranes were blocked with 5% dried non-fat milk for 1 h at room temperature and incubated with the desired primary antibody diluted 1:1000 in blocking buffer overnight at 4 C. Membranes were probed with antibodies to phospho <t>VEGFR2</t> Y996 , phospho VEGFR2 Y1056 , Src Y461 , phospho FAK Y861 and phospho FAK Y925 . Primary antibody incubation was followed by incubation with a horseradish peroxidase-conjugated secondary antibody in blocking buffer for 2 h at room temperature. Proteins were visualized with electrochemiluminescence detection reagents and detected by autoradiography. ( A ) IR-CM effect on phosphorylation of VEGFR2 in U251 glioma cells. ( B ) IR-CM with/without VEGF antibody (10 μg/ml) effect on the phosphorylation of Src and FAK in U251 glioma cells. Blots are representative images of three replicates.
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    Cell Signaling Technology Inc anti akt antibodies
    Growth factor-starved primary HCECs cells were either left unchallenged (CT) or challenged with purified flagellin (250 ng/ml) from indicated bacterial strains; PF(wild type PAK), LF (L94A), QF (Q83A), and then lysed 60 min post-stimulation. Equal amounts of proteins of 30–45 µg were subjected to Western blot using antibodies against phospho- (p) <t>proteins</t> <t>IκB-α,</t> P38, EGFR Y 1173 , ERK or <t>AKT.</t> After stripping off the immunoreactivities, the membranes were reprobed with antibodies against, corresponding proteins for proper protein loading. The Figure is a representative of three independent experiments.
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    Cell Signaling Technology Inc p85
    Effect of BAY on the activation of upstream signaling for NF- κ B translocation. (a to c) RAW264.7 cells (a left panel, b, and c) or peritoneal macrophages (a right panel) (5 × 10 6 cells/mL) were incubated with BAY (15 μ M) in the presence or absence of LPS (1 μ g/mL) for indicated times. After preparing whole lysates, levels of phospho (p)- or total proteins of I κ B α , IKK α / β , Akt, <t>p85/PI3K,</t> Syk, and Src were identified by immunoprecipitation. Relative intensity (a right panel) was calculated by densitometric scanning.
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    Cell Signaling Technology Inc anti akt
    ( A ) L6 myotubes were incubated with serum-free media containing 2% (w/v) BSA (vehicle) ± 700 μM (A) oleic acid (OA) or (B) linoleic acid (LOA) for 16h prior to stimulation with insulin at concentrations indicated (10min). ( C, D ) L6 myotubes were incubated as in (A) prior to stimulation with insulin (20 nM, 10min). ( E ) Human myotubes were incubated with 200 μM OA or LOA for 24h prior to stimulation with insulin (20 nM, 10min). Cell lysates from A-E were subsequently immunoblotted using antibodies against the proteins indicated with quantification of <t>phospho-Akt</t> (Ser473 and Thr308) <t>and</t> <t>phospho-ERK1/2</t> shown in panel A,B and D expressed as mean ± SEM of at least three experiments. The immunoblots shown in panel E are representative of two separate experiments. The asterisks signify a significant change (p<0.05) between the indicated bars (A,B) or the insulin-treatment alone (D).
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    Image Search Results


    The effects of irradiated conditioned medium (IR-CM) on activation of kinases in glioma cells . After 16 h incubation of GBM glioma cells with IR-CM ± monoclonal anti-human VEGF 165 antibody (R&D System), cells were harvested, processed to make lysates and the supernatant was collected. Equal quantities of protein were separated by SDS/PAGE and transferred to PVDF membrane. The membranes were blocked with 5% dried non-fat milk for 1 h at room temperature and incubated with the desired primary antibody diluted 1:1000 in blocking buffer overnight at 4 C. Membranes were probed with antibodies to phospho VEGFR2 Y996 , phospho VEGFR2 Y1056 , Src Y461 , phospho FAK Y861 and phospho FAK Y925 . Primary antibody incubation was followed by incubation with a horseradish peroxidase-conjugated secondary antibody in blocking buffer for 2 h at room temperature. Proteins were visualized with electrochemiluminescence detection reagents and detected by autoradiography. ( A ) IR-CM effect on phosphorylation of VEGFR2 in U251 glioma cells. ( B ) IR-CM with/without VEGF antibody (10 μg/ml) effect on the phosphorylation of Src and FAK in U251 glioma cells. Blots are representative images of three replicates.

    Journal: Radiation Oncology (London, England)

    Article Title: Post-radiation increase in VEGF enhances glioma cell motility in vitro

    doi: 10.1186/1748-717X-7-25

    Figure Lengend Snippet: The effects of irradiated conditioned medium (IR-CM) on activation of kinases in glioma cells . After 16 h incubation of GBM glioma cells with IR-CM ± monoclonal anti-human VEGF 165 antibody (R&D System), cells were harvested, processed to make lysates and the supernatant was collected. Equal quantities of protein were separated by SDS/PAGE and transferred to PVDF membrane. The membranes were blocked with 5% dried non-fat milk for 1 h at room temperature and incubated with the desired primary antibody diluted 1:1000 in blocking buffer overnight at 4 C. Membranes were probed with antibodies to phospho VEGFR2 Y996 , phospho VEGFR2 Y1056 , Src Y461 , phospho FAK Y861 and phospho FAK Y925 . Primary antibody incubation was followed by incubation with a horseradish peroxidase-conjugated secondary antibody in blocking buffer for 2 h at room temperature. Proteins were visualized with electrochemiluminescence detection reagents and detected by autoradiography. ( A ) IR-CM effect on phosphorylation of VEGFR2 in U251 glioma cells. ( B ) IR-CM with/without VEGF antibody (10 μg/ml) effect on the phosphorylation of Src and FAK in U251 glioma cells. Blots are representative images of three replicates.

    Article Snippet: The phosphospecific rabbit antibodies against FAK at Y861 and Y925 were from Millipore (Temecula, CA) and Cell Signaling Technology (Danvers, MA), respectively, and VEGFR2 at Y996 and Y1059 from Cell Signaling Technology.

    Techniques: Irradiation, Activation Assay, Incubation, SDS Page, Blocking Assay, Electrochemiluminescence, Autoradiography

    Growth factor-starved primary HCECs cells were either left unchallenged (CT) or challenged with purified flagellin (250 ng/ml) from indicated bacterial strains; PF(wild type PAK), LF (L94A), QF (Q83A), and then lysed 60 min post-stimulation. Equal amounts of proteins of 30–45 µg were subjected to Western blot using antibodies against phospho- (p) proteins IκB-α, P38, EGFR Y 1173 , ERK or AKT. After stripping off the immunoreactivities, the membranes were reprobed with antibodies against, corresponding proteins for proper protein loading. The Figure is a representative of three independent experiments.

    Journal: PLoS ONE

    Article Title: Flagellin-Induced Corneal Antimicrobial Peptide Production and Wound Repair Involve a Novel NF-κB–Independent and EGFR-Dependent Pathway

    doi: 10.1371/journal.pone.0009351

    Figure Lengend Snippet: Growth factor-starved primary HCECs cells were either left unchallenged (CT) or challenged with purified flagellin (250 ng/ml) from indicated bacterial strains; PF(wild type PAK), LF (L94A), QF (Q83A), and then lysed 60 min post-stimulation. Equal amounts of proteins of 30–45 µg were subjected to Western blot using antibodies against phospho- (p) proteins IκB-α, P38, EGFR Y 1173 , ERK or AKT. After stripping off the immunoreactivities, the membranes were reprobed with antibodies against, corresponding proteins for proper protein loading. The Figure is a representative of three independent experiments.

    Article Snippet: Anti-phospho-IκB-α, anti-IκB-α, anti-phospho-p38, anti-p38, anti-phospho-JNK, anti-JNK, anti-phospho-AKT and anti-AKT antibodies were purchased from Cell Signaling Technology; anti-phospho-ERK1/2, anti-ERK2, anti-phospho-EGFR, anti-EGFR, and anti-hBD-2 antibodies from SantaCruz Biotechnology; TGF-α neutralizing antibody from Oncogen; anti-LL-37 antibody from PANATecs.

    Techniques: Purification, Western Blot, Stripping Membranes

    Growth factor-starved HUCL cells were pretreated with AG1478 (AG, 1 µM), CRM197 (CRM, 10 ng/µl), or TGF-α neutralizing antibody (α-TGF-α 5 µg/ml) for 1 h and then challenged with purified wild type PAK flagellin (PF). One hour post flagellin challenge, cells were lysed and equal amounts of proteins of 30–45 µg were subjected to Western blot using antibodies against phospho- (p) proteins JNK, EGFR at Y 1173 , ERK or AKT. After stripping off the immunoreactivities, the membranes were reprobed with antibodies against proteins for proper loading. The Figure is a representative of two independent experiments.

    Journal: PLoS ONE

    Article Title: Flagellin-Induced Corneal Antimicrobial Peptide Production and Wound Repair Involve a Novel NF-κB–Independent and EGFR-Dependent Pathway

    doi: 10.1371/journal.pone.0009351

    Figure Lengend Snippet: Growth factor-starved HUCL cells were pretreated with AG1478 (AG, 1 µM), CRM197 (CRM, 10 ng/µl), or TGF-α neutralizing antibody (α-TGF-α 5 µg/ml) for 1 h and then challenged with purified wild type PAK flagellin (PF). One hour post flagellin challenge, cells were lysed and equal amounts of proteins of 30–45 µg were subjected to Western blot using antibodies against phospho- (p) proteins JNK, EGFR at Y 1173 , ERK or AKT. After stripping off the immunoreactivities, the membranes were reprobed with antibodies against proteins for proper loading. The Figure is a representative of two independent experiments.

    Article Snippet: Anti-phospho-IκB-α, anti-IκB-α, anti-phospho-p38, anti-p38, anti-phospho-JNK, anti-JNK, anti-phospho-AKT and anti-AKT antibodies were purchased from Cell Signaling Technology; anti-phospho-ERK1/2, anti-ERK2, anti-phospho-EGFR, anti-EGFR, and anti-hBD-2 antibodies from SantaCruz Biotechnology; TGF-α neutralizing antibody from Oncogen; anti-LL-37 antibody from PANATecs.

    Techniques: Purification, Western Blot, Stripping Membranes

    HUCL cells were either pretreated with 50 ng/ml PAK (PF) or L94A (LF) flagellin or left untreated (C) for 24 h and then challenged with (PAK) or without (Control) live PAK for 1 h. Total cell lysates were blotted with antibodies specific for phospho-ERK, AKT, IκB-α, and P38, followed by reprobing with antibodies against corresponding proteins. The data shown are representative of 2 independent experiments.

    Journal: PLoS ONE

    Article Title: Flagellin-Induced Corneal Antimicrobial Peptide Production and Wound Repair Involve a Novel NF-κB–Independent and EGFR-Dependent Pathway

    doi: 10.1371/journal.pone.0009351

    Figure Lengend Snippet: HUCL cells were either pretreated with 50 ng/ml PAK (PF) or L94A (LF) flagellin or left untreated (C) for 24 h and then challenged with (PAK) or without (Control) live PAK for 1 h. Total cell lysates were blotted with antibodies specific for phospho-ERK, AKT, IκB-α, and P38, followed by reprobing with antibodies against corresponding proteins. The data shown are representative of 2 independent experiments.

    Article Snippet: Anti-phospho-IκB-α, anti-IκB-α, anti-phospho-p38, anti-p38, anti-phospho-JNK, anti-JNK, anti-phospho-AKT and anti-AKT antibodies were purchased from Cell Signaling Technology; anti-phospho-ERK1/2, anti-ERK2, anti-phospho-EGFR, anti-EGFR, and anti-hBD-2 antibodies from SantaCruz Biotechnology; TGF-α neutralizing antibody from Oncogen; anti-LL-37 antibody from PANATecs.

    Techniques:

    Epithelial cells in healing porcine corneas treated with wild type (PF), L94A (LF) flagellin, or medium alone (CT), were collected 48 hrs post wounding, lysed in RIPA buffer and sonicated. Equal amounts of cell lysates from 2 of 5 corneas (randomly picked) from each group were immunoblotted with antibodies against phospho-EGFR, AKT or ERK1/2. The immunoreactivities were stripped off and the same membranes were reprobed with antibodies against cellular EGFR, AKT or ERK2, respectively, for proper protein loading.

    Journal: PLoS ONE

    Article Title: Flagellin-Induced Corneal Antimicrobial Peptide Production and Wound Repair Involve a Novel NF-κB–Independent and EGFR-Dependent Pathway

    doi: 10.1371/journal.pone.0009351

    Figure Lengend Snippet: Epithelial cells in healing porcine corneas treated with wild type (PF), L94A (LF) flagellin, or medium alone (CT), were collected 48 hrs post wounding, lysed in RIPA buffer and sonicated. Equal amounts of cell lysates from 2 of 5 corneas (randomly picked) from each group were immunoblotted with antibodies against phospho-EGFR, AKT or ERK1/2. The immunoreactivities were stripped off and the same membranes were reprobed with antibodies against cellular EGFR, AKT or ERK2, respectively, for proper protein loading.

    Article Snippet: Anti-phospho-IκB-α, anti-IκB-α, anti-phospho-p38, anti-p38, anti-phospho-JNK, anti-JNK, anti-phospho-AKT and anti-AKT antibodies were purchased from Cell Signaling Technology; anti-phospho-ERK1/2, anti-ERK2, anti-phospho-EGFR, anti-EGFR, and anti-hBD-2 antibodies from SantaCruz Biotechnology; TGF-α neutralizing antibody from Oncogen; anti-LL-37 antibody from PANATecs.

    Techniques: Sonication

    Effect of BAY on the activation of upstream signaling for NF- κ B translocation. (a to c) RAW264.7 cells (a left panel, b, and c) or peritoneal macrophages (a right panel) (5 × 10 6 cells/mL) were incubated with BAY (15 μ M) in the presence or absence of LPS (1 μ g/mL) for indicated times. After preparing whole lysates, levels of phospho (p)- or total proteins of I κ B α , IKK α / β , Akt, p85/PI3K, Syk, and Src were identified by immunoprecipitation. Relative intensity (a right panel) was calculated by densitometric scanning.

    Journal: Mediators of Inflammation

    Article Title: BAY 11-7082 Is a Broad-Spectrum Inhibitor with Anti-Inflammatory Activity against Multiple Targets

    doi: 10.1155/2012/416036

    Figure Lengend Snippet: Effect of BAY on the activation of upstream signaling for NF- κ B translocation. (a to c) RAW264.7 cells (a left panel, b, and c) or peritoneal macrophages (a right panel) (5 × 10 6 cells/mL) were incubated with BAY (15 μ M) in the presence or absence of LPS (1 μ g/mL) for indicated times. After preparing whole lysates, levels of phospho (p)- or total proteins of I κ B α , IKK α / β , Akt, p85/PI3K, Syk, and Src were identified by immunoprecipitation. Relative intensity (a right panel) was calculated by densitometric scanning.

    Article Snippet: Phosphospecific or total antibodies to p65, p50, Src, Syk, PDK1, p85, Akt, I κ B α , p38, ERK, JNK, lamin A/C, and β actin were from cell signaling (Beverly, MA, USA).

    Techniques: Activation Assay, Translocation Assay, Incubation, Immunoprecipitation

    ( A ) L6 myotubes were incubated with serum-free media containing 2% (w/v) BSA (vehicle) ± 700 μM (A) oleic acid (OA) or (B) linoleic acid (LOA) for 16h prior to stimulation with insulin at concentrations indicated (10min). ( C, D ) L6 myotubes were incubated as in (A) prior to stimulation with insulin (20 nM, 10min). ( E ) Human myotubes were incubated with 200 μM OA or LOA for 24h prior to stimulation with insulin (20 nM, 10min). Cell lysates from A-E were subsequently immunoblotted using antibodies against the proteins indicated with quantification of phospho-Akt (Ser473 and Thr308) and phospho-ERK1/2 shown in panel A,B and D expressed as mean ± SEM of at least three experiments. The immunoblots shown in panel E are representative of two separate experiments. The asterisks signify a significant change (p<0.05) between the indicated bars (A,B) or the insulin-treatment alone (D).

    Journal: PLoS ONE

    Article Title: Enhanced Insulin Sensitivity Associated with Provision of Mono and Polyunsaturated Fatty Acids in Skeletal Muscle Cells Involves Counter Modulation of PP2A

    doi: 10.1371/journal.pone.0092255

    Figure Lengend Snippet: ( A ) L6 myotubes were incubated with serum-free media containing 2% (w/v) BSA (vehicle) ± 700 μM (A) oleic acid (OA) or (B) linoleic acid (LOA) for 16h prior to stimulation with insulin at concentrations indicated (10min). ( C, D ) L6 myotubes were incubated as in (A) prior to stimulation with insulin (20 nM, 10min). ( E ) Human myotubes were incubated with 200 μM OA or LOA for 24h prior to stimulation with insulin (20 nM, 10min). Cell lysates from A-E were subsequently immunoblotted using antibodies against the proteins indicated with quantification of phospho-Akt (Ser473 and Thr308) and phospho-ERK1/2 shown in panel A,B and D expressed as mean ± SEM of at least three experiments. The immunoblots shown in panel E are representative of two separate experiments. The asterisks signify a significant change (p<0.05) between the indicated bars (A,B) or the insulin-treatment alone (D).

    Article Snippet: Phospho-Akt Thr308 , phospho-Akt Ser473 , phospho-ERK1/2 Thr202/Tyr204 , phospho-GSK3 Ser21/9 , phospho-CREB Ser133 , phospho-Src Tyr416 , anti-Akt and anti-ERK1/2 antibodies were from Cell Signaling Technology.

    Techniques: Incubation, Western Blot

    The experimental approach used to test the effect of OA and LOA provision on Akt and ERK1/2 phosphorylation is depicted in (A). L6 myotubes were incubated with serum-free media containing 2% (w/v) BSA (vehicle) ± 700 μM (B and C) oleic acid (OA) or (D and E) linoleic acid (LOA) for 16h prior to stimulation with insulin (20 nM, 10min). Cells were washed to remove insulin and subsequently incubated with 100 nM wortmannin (WM) and lysed at times indicated. Immunoblots and relevant quantification of phospho-Akt and phospho-ERK1/2 in L6 myotubes incubated with OA (B,C) or LOA (D,E). Data in the bar graphs is presented as mean ± SEM (n = 3) with asterisks indicating a significant change (p<0.05) between the respective filled (fatty acid treatment) and unfilled (control) bar at each time point.

    Journal: PLoS ONE

    Article Title: Enhanced Insulin Sensitivity Associated with Provision of Mono and Polyunsaturated Fatty Acids in Skeletal Muscle Cells Involves Counter Modulation of PP2A

    doi: 10.1371/journal.pone.0092255

    Figure Lengend Snippet: The experimental approach used to test the effect of OA and LOA provision on Akt and ERK1/2 phosphorylation is depicted in (A). L6 myotubes were incubated with serum-free media containing 2% (w/v) BSA (vehicle) ± 700 μM (B and C) oleic acid (OA) or (D and E) linoleic acid (LOA) for 16h prior to stimulation with insulin (20 nM, 10min). Cells were washed to remove insulin and subsequently incubated with 100 nM wortmannin (WM) and lysed at times indicated. Immunoblots and relevant quantification of phospho-Akt and phospho-ERK1/2 in L6 myotubes incubated with OA (B,C) or LOA (D,E). Data in the bar graphs is presented as mean ± SEM (n = 3) with asterisks indicating a significant change (p<0.05) between the respective filled (fatty acid treatment) and unfilled (control) bar at each time point.

    Article Snippet: Phospho-Akt Thr308 , phospho-Akt Ser473 , phospho-ERK1/2 Thr202/Tyr204 , phospho-GSK3 Ser21/9 , phospho-CREB Ser133 , phospho-Src Tyr416 , anti-Akt and anti-ERK1/2 antibodies were from Cell Signaling Technology.

    Techniques: Incubation, Western Blot