sodium orthovanada  (Millipore)


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    Name:
    Sodium orthovanadate
    Description:

    Catalog Number:
    s6508
    Price:
    None
    Applications:
    Sodium orthovandate was used to prepare the stop solution in dephosphorylation assay of insulin receptor kinase.26 It was one of the reagents used in the development of Matrix ChIP that utilizes surface-immobilized antibodies.27
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    Structured Review

    Millipore sodium orthovanada
    Sodium orthovanadate

    https://www.bioz.com/result/sodium orthovanada/product/Millipore
    Average 99 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    sodium orthovanada - by Bioz Stars, 2020-09
    99/100 stars

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    Incubation:

    Article Title: Cyclic trans-phosphorylation in a homodimer as the predominant mechanism of EGFRvIII action and regulation
    Article Snippet: .. To analyse the effects of pharmacological inhibitors, cells were seeded in 6-well plates at 2.5x105 cells per well, incubated overnight to allow for adhesion and serum starved for subsequent 24 h. Serum free medium containing appropriate inhibitors was used to pre-treat cells for 30 min, unless indicated otherwise, prior to stimulation with 20 ng/mL EGF (Sigma-Aldrich, St Louis, MO, USA, Cat. no. E5036), 1 mM sodium orthovanadate – NaOVa (Na3 VO4 , Calbiochem, Cat. no. 567540), phenylarsine oxide – PAO (Sigma, Cat. no. P3075) or pervanadate – pV (obtained as described previously [ ]; using Catalase, Sigma, Cat. no. C1345 and H2 O2 Sigma, Cat. no. 31642) for 1 hour and lysed. .. Following concentrations of inhibitors were used: DMSO (Sigma-Aldrich, Cat. no. D2438, solvent control), 10 μM erlotinib (Tarceva, Molecula; Cat. no. 89983631), 0.5 μM afatinib (Selleckchem, Cat. no. S1011); 5 μM gefitinib (CellSignaling; 4765), 5 μM lapatinib (Selleckchem, Cat. no. S1028), 300 nM flavopiridol (Selleckchem, Cat. no. S1230), 5 μM arry380 (Gentaur Europe, Cat. no. A8366), 10 μM Nu7441 (Selleckchem, Cat. no. S2638) and 10 μM ruxolitinib (Selleckchem, Cat. no. S1378).

    Article Title: A single residue, arginine 65, is critical for the functional interaction of leukocyte associated inhibitory receptor (LAIR)-1 with collagens 1
    Article Snippet: .. LAIR-1 wt or LAIR-1 R65K transfected K562 cells were treated with collagen type I at 1 μg/107 cells/ml in medium without serum at 37 °C for 20 min. For pervanadate treatment, cells were incubated for 20 min with freshly prepared sodium pervanadate (0.1 mM sodium orthovanadate and 10 mM hydrogen peroxide from Sigma-Aldrich) in medium without serum at 37 °C. ..

    Article Title: Cyclic trans-phosphorylation in a homodimer as the predominant mechanism of EGFRvIII action and regulation
    Article Snippet: .. Chemicals To analyse the effects of pharmacological inhibitors, cells were seeded in 6-well plates at 2.5x105 cells per well, incubated overnight to allow for adhesion and serum starved for subsequent 24 h. Serum free medium containing appropriate inhibitors was used to pre-treat cells for 30 min, unless indicated otherwise, prior to stimulation with 20 ng/mL EGF (Sigma-Aldrich, St Louis, MO, USA, Cat. no. E5036), 1 mM sodium orthovanadate – NaOVa (Na3 VO4 , Calbiochem, Cat. no. 567540), phenylarsine oxide – PAO (Sigma, Cat. no. P3075) or pervanadate – pV (obtained as described previously [ ]; using Catalase, Sigma, Cat. no. C1345 and H2 O2 Sigma, Cat. no. 31642) for 1 hour and lysed. .. Following concentrations of inhibitors were used: DMSO (Sigma-Aldrich, Cat. no. D2438, solvent control), 10 μM erlotinib (Tarceva, Molecula; Cat. no. 89983631), 0.5 μM afatinib (Selleckchem, Cat. no. S1011); 5 μM gefitinib (CellSignaling; 4765), 5 μM lapatinib (Selleckchem, Cat. no. S1028), 300 nM flavopiridol (Selleckchem, Cat. no. S1230), 5 μM arry380 (Gentaur Europe, Cat. no. A8366), 10 μM Nu7441 (Selleckchem, Cat. no. S2638) and 10 μM ruxolitinib (Selleckchem, Cat. no. S1378).

    other:

    Article Title: Hypoxia promotes chemoresistance in acute lymphoblastic leukemia cell lines by modulating death signaling pathways
    Article Snippet: Briefly, RPPA assay was performed on 1 mg/mL of protein lysed with 20 mM of Hepes (pH7.9, Sigma-Aldrich®), 1 mM of MgCl2 (Sigma-Aldrich®), 1 % of NP-40 substitute (VWR®, Fontenay-Sous-Bois, France), 0.5 % of Sodium cholate (Sigma-Aldrich®), 0.25 % of n-dodecyl-β-D-maltoside (VWR®), 1 mM of Sodium orthovanadate (Sigma-Aldrich®) and 50 mM of Sodium fluoride (Sigma-Aldrich®) containing freshly added protease inhibitors and phosphatase inhibitors (Fisher Scientifics®, Illkirch, France).

    Transfection:

    Article Title: A single residue, arginine 65, is critical for the functional interaction of leukocyte associated inhibitory receptor (LAIR)-1 with collagens 1
    Article Snippet: .. LAIR-1 wt or LAIR-1 R65K transfected K562 cells were treated with collagen type I at 1 μg/107 cells/ml in medium without serum at 37 °C for 20 min. For pervanadate treatment, cells were incubated for 20 min with freshly prepared sodium pervanadate (0.1 mM sodium orthovanadate and 10 mM hydrogen peroxide from Sigma-Aldrich) in medium without serum at 37 °C. ..

    Positive Control:

    Article Title: Activation of Triggering Receptor Expressed on Myeloid Cells-1 on Human Neutrophils by Marburg and Ebola Viruses
    Article Snippet: .. Pervanadate (sodium orthovanadate plus H2 O2 ; Sigma, St. Louis, MO) was used as a positive control ( ). .. After stimulation, neutrophils were lysed and lysates were immune precipitated using polyclonal anti-DAP12 antibody (kindly provided by D. McVicar, NCI, Frederick, MD), separated on a 10% Bis-Tris NuPAGE gel (Invitrogen, Carlsbad, CA), and transferred onto activated polyvinylidene difluoride membranes (Millipore, Billerica, MA).

    Article Title: Aberrant STAT5 and PI3K/mTOR pathway signaling occurs in human CRLF2-rearranged B-precursor acute lymphoblastic leukemia
    Article Snippet: .. Pervanadate (125μM; prepared from sodium orthovanadate and 3% hydrogen peroxide; Sigma-Aldrich), an irreversible protein tyrosine phosphatase inhibitor, was also used as a positive control to elicit maximal phosphorylation of each signaling protein. .. Cells were immediately lysed in NP-40 cell lysis buffer (Invitrogen) supplemented with 1% protease and phosphatase inhibitors (Calbiochem).

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  • 99
    Millipore sodium orthovanadate naova
    Treatment with <t>EGF</t> does not affect phosphorylation of EGFRvIII ( A ) DK-MG cells treated with indicated concentration of EGF for 10 min were lysed and analysed by Western blotting. ( B ) Quantification of EGFRvIII phosphorylation as shown in A. Statistical analysis performed using one-way ANOVA with post-analysis Bonferroni’s multiple comparisons test. ( C ) Western blot analysis of cells stimulated for 1 h with 20 ng/mL EGF, 1 mM <t>NaOVa,</t> concomitant EGF and NaOVa or Control cells, as indicated. ( D ) Quantification of blots as shown in C, with normalization to wild-type receptor under Control conditions. Statistical analysis performed using two-way ANOVA with post-analysis Bonferroni’s multiple comparisons test. *** p
    Sodium Orthovanadate Naova, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sodium orthovanadate naova/product/Millipore
    Average 99 stars, based on 1113 article reviews
    Price from $9.99 to $1999.99
    sodium orthovanadate naova - by Bioz Stars, 2020-09
    99/100 stars
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    99
    Millipore egfr ub lysis buffer
    Epidermal growth factor–dependent ubiquitylation of the <t>EGFR</t> in primary and immortalized corneal epithelial cells. ( A ) Primary human corneal epithelial cells and immortalized corneal epithelial cells (hTCEpi) were treated without (−) or
    Egfr Ub Lysis Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egfr ub lysis buffer/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Treatment with EGF does not affect phosphorylation of EGFRvIII ( A ) DK-MG cells treated with indicated concentration of EGF for 10 min were lysed and analysed by Western blotting. ( B ) Quantification of EGFRvIII phosphorylation as shown in A. Statistical analysis performed using one-way ANOVA with post-analysis Bonferroni’s multiple comparisons test. ( C ) Western blot analysis of cells stimulated for 1 h with 20 ng/mL EGF, 1 mM NaOVa, concomitant EGF and NaOVa or Control cells, as indicated. ( D ) Quantification of blots as shown in C, with normalization to wild-type receptor under Control conditions. Statistical analysis performed using two-way ANOVA with post-analysis Bonferroni’s multiple comparisons test. *** p

    Journal: Oncotarget

    Article Title: Cyclic trans-phosphorylation in a homodimer as the predominant mechanism of EGFRvIII action and regulation

    doi: 10.18632/oncotarget.24058

    Figure Lengend Snippet: Treatment with EGF does not affect phosphorylation of EGFRvIII ( A ) DK-MG cells treated with indicated concentration of EGF for 10 min were lysed and analysed by Western blotting. ( B ) Quantification of EGFRvIII phosphorylation as shown in A. Statistical analysis performed using one-way ANOVA with post-analysis Bonferroni’s multiple comparisons test. ( C ) Western blot analysis of cells stimulated for 1 h with 20 ng/mL EGF, 1 mM NaOVa, concomitant EGF and NaOVa or Control cells, as indicated. ( D ) Quantification of blots as shown in C, with normalization to wild-type receptor under Control conditions. Statistical analysis performed using two-way ANOVA with post-analysis Bonferroni’s multiple comparisons test. *** p

    Article Snippet: Chemicals To analyse the effects of pharmacological inhibitors, cells were seeded in 6-well plates at 2.5x105 cells per well, incubated overnight to allow for adhesion and serum starved for subsequent 24 h. Serum free medium containing appropriate inhibitors was used to pre-treat cells for 30 min, unless indicated otherwise, prior to stimulation with 20 ng/mL EGF (Sigma-Aldrich, St Louis, MO, USA, Cat. no. E5036), 1 mM sodium orthovanadate – NaOVa (Na3 VO4 , Calbiochem, Cat. no. 567540), phenylarsine oxide – PAO (Sigma, Cat. no. P3075) or pervanadate – pV (obtained as described previously [ ]; using Catalase, Sigma, Cat. no. C1345 and H2 O2 Sigma, Cat. no. 31642) for 1 hour and lysed.

    Techniques: Concentration Assay, Western Blot

    Homodimerization of EGFRvIII ( A ) Semi-native Western blot (non-reducing conditions) analysis of the AD293 cell lines expressing EGFRvIII or EGFRvIIIC16S treated simultaneously with EGFR TKIs and NaOVa, as indicated. Arrowheads indicate dimers, arrows point to monomers. ( B ) DK-MG low , cell line with marginal endogenous EGFRvIII expression, were transduced to constitutively express either naïve EGFRvIII (DK-MG exo ) or C16S mutated version (DK-MG C16S ). Cells were treated with NaOVa or EGF, as indicated. Immunoblots have been uniformly adjusted for brightness and contrast to facilitate interpretation.

    Journal: Oncotarget

    Article Title: Cyclic trans-phosphorylation in a homodimer as the predominant mechanism of EGFRvIII action and regulation

    doi: 10.18632/oncotarget.24058

    Figure Lengend Snippet: Homodimerization of EGFRvIII ( A ) Semi-native Western blot (non-reducing conditions) analysis of the AD293 cell lines expressing EGFRvIII or EGFRvIIIC16S treated simultaneously with EGFR TKIs and NaOVa, as indicated. Arrowheads indicate dimers, arrows point to monomers. ( B ) DK-MG low , cell line with marginal endogenous EGFRvIII expression, were transduced to constitutively express either naïve EGFRvIII (DK-MG exo ) or C16S mutated version (DK-MG C16S ). Cells were treated with NaOVa or EGF, as indicated. Immunoblots have been uniformly adjusted for brightness and contrast to facilitate interpretation.

    Article Snippet: Chemicals To analyse the effects of pharmacological inhibitors, cells were seeded in 6-well plates at 2.5x105 cells per well, incubated overnight to allow for adhesion and serum starved for subsequent 24 h. Serum free medium containing appropriate inhibitors was used to pre-treat cells for 30 min, unless indicated otherwise, prior to stimulation with 20 ng/mL EGF (Sigma-Aldrich, St Louis, MO, USA, Cat. no. E5036), 1 mM sodium orthovanadate – NaOVa (Na3 VO4 , Calbiochem, Cat. no. 567540), phenylarsine oxide – PAO (Sigma, Cat. no. P3075) or pervanadate – pV (obtained as described previously [ ]; using Catalase, Sigma, Cat. no. C1345 and H2 O2 Sigma, Cat. no. 31642) for 1 hour and lysed.

    Techniques: Western Blot, Expressing

    Treatment of DK-MG cells with phosphatase inhibitors results in hyperphosphorylation of EGFRwt and EGFRvIII on majority of tyrosine residues ( A ) Western blot analysis of EGFR phosphorylation in cells treated with NaOVa, pV or PAO at indicated concentrations for 1 h. ( B ) Analysis of phosphorylation status of EGFR on selected tyrosine residues following stimulation with 20 ng/mL EGF, 1 mM NaOVa or 0.5 µM PAO. ( C–F ) Ratio of phosphorylated tyrosine to the total EGFR protein for the wild-type and mutant protein following stimulation as indicated in B, for residue Tyr 1045 (C), Tyr1068 (D), Tyr1148 (E) and Tyr 1173 (F). Statistical analysis performed using one-way ANOVA with post-analysis Bonferroni’s multiple comparisons test for each residue within one receptor variant. ( G ) Quantification of phosphorylated tyrosine 1068 to total EGFR for EGFRwt and EGFRvIII is presented. Statistical analysis performed using two-way ANOVA with post-analysis Bonferroni’s multiple comparisons test, n = 23. *** p

    Journal: Oncotarget

    Article Title: Cyclic trans-phosphorylation in a homodimer as the predominant mechanism of EGFRvIII action and regulation

    doi: 10.18632/oncotarget.24058

    Figure Lengend Snippet: Treatment of DK-MG cells with phosphatase inhibitors results in hyperphosphorylation of EGFRwt and EGFRvIII on majority of tyrosine residues ( A ) Western blot analysis of EGFR phosphorylation in cells treated with NaOVa, pV or PAO at indicated concentrations for 1 h. ( B ) Analysis of phosphorylation status of EGFR on selected tyrosine residues following stimulation with 20 ng/mL EGF, 1 mM NaOVa or 0.5 µM PAO. ( C–F ) Ratio of phosphorylated tyrosine to the total EGFR protein for the wild-type and mutant protein following stimulation as indicated in B, for residue Tyr 1045 (C), Tyr1068 (D), Tyr1148 (E) and Tyr 1173 (F). Statistical analysis performed using one-way ANOVA with post-analysis Bonferroni’s multiple comparisons test for each residue within one receptor variant. ( G ) Quantification of phosphorylated tyrosine 1068 to total EGFR for EGFRwt and EGFRvIII is presented. Statistical analysis performed using two-way ANOVA with post-analysis Bonferroni’s multiple comparisons test, n = 23. *** p

    Article Snippet: Chemicals To analyse the effects of pharmacological inhibitors, cells were seeded in 6-well plates at 2.5x105 cells per well, incubated overnight to allow for adhesion and serum starved for subsequent 24 h. Serum free medium containing appropriate inhibitors was used to pre-treat cells for 30 min, unless indicated otherwise, prior to stimulation with 20 ng/mL EGF (Sigma-Aldrich, St Louis, MO, USA, Cat. no. E5036), 1 mM sodium orthovanadate – NaOVa (Na3 VO4 , Calbiochem, Cat. no. 567540), phenylarsine oxide – PAO (Sigma, Cat. no. P3075) or pervanadate – pV (obtained as described previously [ ]; using Catalase, Sigma, Cat. no. C1345 and H2 O2 Sigma, Cat. no. 31642) for 1 hour and lysed.

    Techniques: Western Blot, Mutagenesis, Variant Assay

    EGFRvIII is not phosphorylated by EGFRwt ( A ) Western blot analysis of cells treated with indicated inhibitors prior to and concurrently with EGF or NaOVa treatment for 1 h. Concentrations of inhibitors were based on literature reports. ( B ) Cells were treated with inhibitors specific to pan-JAK (ruxolitinib), CDK2 (flavopiridol) or DNA-PK (Nu-7441) prior to and concurrently with NaOVa treatment. Concentrations of inhibitors were based on literature reports. ( C ) Cells transiently expressing control scrambled (SCR) shRNA or shRNA targeted against EGFRwt were treated as indicated and analyzed by Western blotting. ( D ) Quantification of blots as shown in C, with columns representing ratio of phosphorylated to total EGFRvIII protein. ( E ) Cells treated with cycloheximide and stimulated with EGF for 3 h as indicated. Where indicated, cells were incubated for another 30 min with DMSO or gefitinib prior to 1 h treatment with NaOVa. ( F ) Quantification of blots as in E for EGFRvIII, with normalization to DMSO treated Control cells. Statistical analysis performed using two-way ANOVA with post-analysis Bonferroni’s multiple comparisons test. ** p

    Journal: Oncotarget

    Article Title: Cyclic trans-phosphorylation in a homodimer as the predominant mechanism of EGFRvIII action and regulation

    doi: 10.18632/oncotarget.24058

    Figure Lengend Snippet: EGFRvIII is not phosphorylated by EGFRwt ( A ) Western blot analysis of cells treated with indicated inhibitors prior to and concurrently with EGF or NaOVa treatment for 1 h. Concentrations of inhibitors were based on literature reports. ( B ) Cells were treated with inhibitors specific to pan-JAK (ruxolitinib), CDK2 (flavopiridol) or DNA-PK (Nu-7441) prior to and concurrently with NaOVa treatment. Concentrations of inhibitors were based on literature reports. ( C ) Cells transiently expressing control scrambled (SCR) shRNA or shRNA targeted against EGFRwt were treated as indicated and analyzed by Western blotting. ( D ) Quantification of blots as shown in C, with columns representing ratio of phosphorylated to total EGFRvIII protein. ( E ) Cells treated with cycloheximide and stimulated with EGF for 3 h as indicated. Where indicated, cells were incubated for another 30 min with DMSO or gefitinib prior to 1 h treatment with NaOVa. ( F ) Quantification of blots as in E for EGFRvIII, with normalization to DMSO treated Control cells. Statistical analysis performed using two-way ANOVA with post-analysis Bonferroni’s multiple comparisons test. ** p

    Article Snippet: Chemicals To analyse the effects of pharmacological inhibitors, cells were seeded in 6-well plates at 2.5x105 cells per well, incubated overnight to allow for adhesion and serum starved for subsequent 24 h. Serum free medium containing appropriate inhibitors was used to pre-treat cells for 30 min, unless indicated otherwise, prior to stimulation with 20 ng/mL EGF (Sigma-Aldrich, St Louis, MO, USA, Cat. no. E5036), 1 mM sodium orthovanadate – NaOVa (Na3 VO4 , Calbiochem, Cat. no. 567540), phenylarsine oxide – PAO (Sigma, Cat. no. P3075) or pervanadate – pV (obtained as described previously [ ]; using Catalase, Sigma, Cat. no. C1345 and H2 O2 Sigma, Cat. no. 31642) for 1 hour and lysed.

    Techniques: Western Blot, Expressing, shRNA, Incubation

    EGFRwt is dispensable for EGFRvIII phosphorylation ( A ) AD293 cell lines stably expressing EGFRvIII was treated with erlotinib prior to and simultaneously with NaOVa stimulation. ( B ) AD293 cell line expressing either naïve or kinase dead (KD) version of EGFRvIII on its own or together with EGFRwt have been treated with EGF or NaOVa, as indicated.

    Journal: Oncotarget

    Article Title: Cyclic trans-phosphorylation in a homodimer as the predominant mechanism of EGFRvIII action and regulation

    doi: 10.18632/oncotarget.24058

    Figure Lengend Snippet: EGFRwt is dispensable for EGFRvIII phosphorylation ( A ) AD293 cell lines stably expressing EGFRvIII was treated with erlotinib prior to and simultaneously with NaOVa stimulation. ( B ) AD293 cell line expressing either naïve or kinase dead (KD) version of EGFRvIII on its own or together with EGFRwt have been treated with EGF or NaOVa, as indicated.

    Article Snippet: Chemicals To analyse the effects of pharmacological inhibitors, cells were seeded in 6-well plates at 2.5x105 cells per well, incubated overnight to allow for adhesion and serum starved for subsequent 24 h. Serum free medium containing appropriate inhibitors was used to pre-treat cells for 30 min, unless indicated otherwise, prior to stimulation with 20 ng/mL EGF (Sigma-Aldrich, St Louis, MO, USA, Cat. no. E5036), 1 mM sodium orthovanadate – NaOVa (Na3 VO4 , Calbiochem, Cat. no. 567540), phenylarsine oxide – PAO (Sigma, Cat. no. P3075) or pervanadate – pV (obtained as described previously [ ]; using Catalase, Sigma, Cat. no. C1345 and H2 O2 Sigma, Cat. no. 31642) for 1 hour and lysed.

    Techniques: Stable Transfection, Expressing

    Treatment with EGF does not affect phosphorylation of EGFRvIII ( A ) DK-MG cells treated with indicated concentration of EGF for 10 min were lysed and analysed by Western blotting. ( B ) Quantification of EGFRvIII phosphorylation as shown in A. Statistical analysis performed using one-way ANOVA with post-analysis Bonferroni’s multiple comparisons test. ( C ) Western blot analysis of cells stimulated for 1 h with 20 ng/mL EGF, 1 mM NaOVa, concomitant EGF and NaOVa or Control cells, as indicated. ( D ) Quantification of blots as shown in C, with normalization to wild-type receptor under Control conditions. Statistical analysis performed using two-way ANOVA with post-analysis Bonferroni’s multiple comparisons test. *** p

    Journal: Oncotarget

    Article Title: Cyclic trans-phosphorylation in a homodimer as the predominant mechanism of EGFRvIII action and regulation

    doi: 10.18632/oncotarget.24058

    Figure Lengend Snippet: Treatment with EGF does not affect phosphorylation of EGFRvIII ( A ) DK-MG cells treated with indicated concentration of EGF for 10 min were lysed and analysed by Western blotting. ( B ) Quantification of EGFRvIII phosphorylation as shown in A. Statistical analysis performed using one-way ANOVA with post-analysis Bonferroni’s multiple comparisons test. ( C ) Western blot analysis of cells stimulated for 1 h with 20 ng/mL EGF, 1 mM NaOVa, concomitant EGF and NaOVa or Control cells, as indicated. ( D ) Quantification of blots as shown in C, with normalization to wild-type receptor under Control conditions. Statistical analysis performed using two-way ANOVA with post-analysis Bonferroni’s multiple comparisons test. *** p

    Article Snippet: To analyse the effects of pharmacological inhibitors, cells were seeded in 6-well plates at 2.5x105 cells per well, incubated overnight to allow for adhesion and serum starved for subsequent 24 h. Serum free medium containing appropriate inhibitors was used to pre-treat cells for 30 min, unless indicated otherwise, prior to stimulation with 20 ng/mL EGF (Sigma-Aldrich, St Louis, MO, USA, Cat. no. E5036), 1 mM sodium orthovanadate – NaOVa (Na3 VO4 , Calbiochem, Cat. no. 567540), phenylarsine oxide – PAO (Sigma, Cat. no. P3075) or pervanadate – pV (obtained as described previously [ ]; using Catalase, Sigma, Cat. no. C1345 and H2 O2 Sigma, Cat. no. 31642) for 1 hour and lysed.

    Techniques: Concentration Assay, Western Blot

    EGFRvIII is not phosphorylated by EGFRwt ( A ) Western blot analysis of cells treated with indicated inhibitors prior to and concurrently with EGF or NaOVa treatment for 1 h. Concentrations of inhibitors were based on literature reports. ( B ) Cells were treated with inhibitors specific to pan-JAK (ruxolitinib), CDK2 (flavopiridol) or DNA-PK (Nu-7441) prior to and concurrently with NaOVa treatment. Concentrations of inhibitors were based on literature reports. ( C ) Cells transiently expressing control scrambled (SCR) shRNA or shRNA targeted against EGFRwt were treated as indicated and analyzed by Western blotting. ( D ) Quantification of blots as shown in C, with columns representing ratio of phosphorylated to total EGFRvIII protein. ( E ) Cells treated with cycloheximide and stimulated with EGF for 3 h as indicated. Where indicated, cells were incubated for another 30 min with DMSO or gefitinib prior to 1 h treatment with NaOVa. ( F ) Quantification of blots as in E for EGFRvIII, with normalization to DMSO treated Control cells. Statistical analysis performed using two-way ANOVA with post-analysis Bonferroni’s multiple comparisons test. ** p

    Journal: Oncotarget

    Article Title: Cyclic trans-phosphorylation in a homodimer as the predominant mechanism of EGFRvIII action and regulation

    doi: 10.18632/oncotarget.24058

    Figure Lengend Snippet: EGFRvIII is not phosphorylated by EGFRwt ( A ) Western blot analysis of cells treated with indicated inhibitors prior to and concurrently with EGF or NaOVa treatment for 1 h. Concentrations of inhibitors were based on literature reports. ( B ) Cells were treated with inhibitors specific to pan-JAK (ruxolitinib), CDK2 (flavopiridol) or DNA-PK (Nu-7441) prior to and concurrently with NaOVa treatment. Concentrations of inhibitors were based on literature reports. ( C ) Cells transiently expressing control scrambled (SCR) shRNA or shRNA targeted against EGFRwt were treated as indicated and analyzed by Western blotting. ( D ) Quantification of blots as shown in C, with columns representing ratio of phosphorylated to total EGFRvIII protein. ( E ) Cells treated with cycloheximide and stimulated with EGF for 3 h as indicated. Where indicated, cells were incubated for another 30 min with DMSO or gefitinib prior to 1 h treatment with NaOVa. ( F ) Quantification of blots as in E for EGFRvIII, with normalization to DMSO treated Control cells. Statistical analysis performed using two-way ANOVA with post-analysis Bonferroni’s multiple comparisons test. ** p

    Article Snippet: To analyse the effects of pharmacological inhibitors, cells were seeded in 6-well plates at 2.5x105 cells per well, incubated overnight to allow for adhesion and serum starved for subsequent 24 h. Serum free medium containing appropriate inhibitors was used to pre-treat cells for 30 min, unless indicated otherwise, prior to stimulation with 20 ng/mL EGF (Sigma-Aldrich, St Louis, MO, USA, Cat. no. E5036), 1 mM sodium orthovanadate – NaOVa (Na3 VO4 , Calbiochem, Cat. no. 567540), phenylarsine oxide – PAO (Sigma, Cat. no. P3075) or pervanadate – pV (obtained as described previously [ ]; using Catalase, Sigma, Cat. no. C1345 and H2 O2 Sigma, Cat. no. 31642) for 1 hour and lysed.

    Techniques: Western Blot, Expressing, shRNA, Incubation

    EGFRwt is dispensable for EGFRvIII phosphorylation ( A ) AD293 cell lines stably expressing EGFRvIII was treated with erlotinib prior to and simultaneously with NaOVa stimulation. ( B ) AD293 cell line expressing either naïve or kinase dead (KD) version of EGFRvIII on its own or together with EGFRwt have been treated with EGF or NaOVa, as indicated.

    Journal: Oncotarget

    Article Title: Cyclic trans-phosphorylation in a homodimer as the predominant mechanism of EGFRvIII action and regulation

    doi: 10.18632/oncotarget.24058

    Figure Lengend Snippet: EGFRwt is dispensable for EGFRvIII phosphorylation ( A ) AD293 cell lines stably expressing EGFRvIII was treated with erlotinib prior to and simultaneously with NaOVa stimulation. ( B ) AD293 cell line expressing either naïve or kinase dead (KD) version of EGFRvIII on its own or together with EGFRwt have been treated with EGF or NaOVa, as indicated.

    Article Snippet: To analyse the effects of pharmacological inhibitors, cells were seeded in 6-well plates at 2.5x105 cells per well, incubated overnight to allow for adhesion and serum starved for subsequent 24 h. Serum free medium containing appropriate inhibitors was used to pre-treat cells for 30 min, unless indicated otherwise, prior to stimulation with 20 ng/mL EGF (Sigma-Aldrich, St Louis, MO, USA, Cat. no. E5036), 1 mM sodium orthovanadate – NaOVa (Na3 VO4 , Calbiochem, Cat. no. 567540), phenylarsine oxide – PAO (Sigma, Cat. no. P3075) or pervanadate – pV (obtained as described previously [ ]; using Catalase, Sigma, Cat. no. C1345 and H2 O2 Sigma, Cat. no. 31642) for 1 hour and lysed.

    Techniques: Stable Transfection, Expressing

    Treatment of DK-MG cells with phosphatase inhibitors results in hyperphosphorylation of EGFRwt and EGFRvIII on majority of tyrosine residues ( A ) Western blot analysis of EGFR phosphorylation in cells treated with NaOVa, pV or PAO at indicated concentrations for 1 h. ( B ) Analysis of phosphorylation status of EGFR on selected tyrosine residues following stimulation with 20 ng/mL EGF, 1 mM NaOVa or 0.5 µM PAO. ( C–F ) Ratio of phosphorylated tyrosine to the total EGFR protein for the wild-type and mutant protein following stimulation as indicated in B, for residue Tyr 1045 (C), Tyr1068 (D), Tyr1148 (E) and Tyr 1173 (F). Statistical analysis performed using one-way ANOVA with post-analysis Bonferroni’s multiple comparisons test for each residue within one receptor variant. ( G ) Quantification of phosphorylated tyrosine 1068 to total EGFR for EGFRwt and EGFRvIII is presented. Statistical analysis performed using two-way ANOVA with post-analysis Bonferroni’s multiple comparisons test, n = 23. *** p

    Journal: Oncotarget

    Article Title: Cyclic trans-phosphorylation in a homodimer as the predominant mechanism of EGFRvIII action and regulation

    doi: 10.18632/oncotarget.24058

    Figure Lengend Snippet: Treatment of DK-MG cells with phosphatase inhibitors results in hyperphosphorylation of EGFRwt and EGFRvIII on majority of tyrosine residues ( A ) Western blot analysis of EGFR phosphorylation in cells treated with NaOVa, pV or PAO at indicated concentrations for 1 h. ( B ) Analysis of phosphorylation status of EGFR on selected tyrosine residues following stimulation with 20 ng/mL EGF, 1 mM NaOVa or 0.5 µM PAO. ( C–F ) Ratio of phosphorylated tyrosine to the total EGFR protein for the wild-type and mutant protein following stimulation as indicated in B, for residue Tyr 1045 (C), Tyr1068 (D), Tyr1148 (E) and Tyr 1173 (F). Statistical analysis performed using one-way ANOVA with post-analysis Bonferroni’s multiple comparisons test for each residue within one receptor variant. ( G ) Quantification of phosphorylated tyrosine 1068 to total EGFR for EGFRwt and EGFRvIII is presented. Statistical analysis performed using two-way ANOVA with post-analysis Bonferroni’s multiple comparisons test, n = 23. *** p

    Article Snippet: To analyse the effects of pharmacological inhibitors, cells were seeded in 6-well plates at 2.5x105 cells per well, incubated overnight to allow for adhesion and serum starved for subsequent 24 h. Serum free medium containing appropriate inhibitors was used to pre-treat cells for 30 min, unless indicated otherwise, prior to stimulation with 20 ng/mL EGF (Sigma-Aldrich, St Louis, MO, USA, Cat. no. E5036), 1 mM sodium orthovanadate – NaOVa (Na3 VO4 , Calbiochem, Cat. no. 567540), phenylarsine oxide – PAO (Sigma, Cat. no. P3075) or pervanadate – pV (obtained as described previously [ ]; using Catalase, Sigma, Cat. no. C1345 and H2 O2 Sigma, Cat. no. 31642) for 1 hour and lysed.

    Techniques: Western Blot, Mutagenesis, Variant Assay

    Epidermal growth factor–dependent ubiquitylation of the EGFR in primary and immortalized corneal epithelial cells. ( A ) Primary human corneal epithelial cells and immortalized corneal epithelial cells (hTCEpi) were treated without (−) or

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Antagonizing c-Cbl Enhances EGFR-Dependent Corneal Epithelial Homeostasis

    doi: 10.1167/iovs.14-14133

    Figure Lengend Snippet: Epidermal growth factor–dependent ubiquitylation of the EGFR in primary and immortalized corneal epithelial cells. ( A ) Primary human corneal epithelial cells and immortalized corneal epithelial cells (hTCEpi) were treated without (−) or

    Article Snippet: Epidermal growth factor receptor ubiquitylation was monitored using a modification of a protocol by Visser Smit et al. Corneal epithelial cells (primary, hTCEpi, or hTCEpi [-c-Cbl]) were serum starved for 2 hours, treated with 100 ng/mL EGF in K-SFM for 10 minutes, and harvested in a chilled EGFR-UB lysis buffer (0.5% Triton x-100/50 mM Tris pH 7.5/150 mM NaCl/1 mM EDTA/1 mM sodium orthovanadate/10 mM sodium fluoride) supplemented with 2 mM phenylmethyl sulfonyl fluoride (PMSF) (Calbiochem, Billerica, MA, USA)/16 μM G5 Ubiquitin isopeptidase inhibitor I (Santa Cruz Biotechnology).

    Techniques:

    The inducible knockdown of c-Cbl promotes EGF-EGFR recycling and enhances corneal epithelial cell migration. Parental hTCEpi ( top ) and hTCEpi (-c-Cbl) ( bottom ) cells were incubated without and with 1 μg/mL doxycycline for 72 hours. ( A ) Recycling

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Antagonizing c-Cbl Enhances EGFR-Dependent Corneal Epithelial Homeostasis

    doi: 10.1167/iovs.14-14133

    Figure Lengend Snippet: The inducible knockdown of c-Cbl promotes EGF-EGFR recycling and enhances corneal epithelial cell migration. Parental hTCEpi ( top ) and hTCEpi (-c-Cbl) ( bottom ) cells were incubated without and with 1 μg/mL doxycycline for 72 hours. ( A ) Recycling

    Article Snippet: Epidermal growth factor receptor ubiquitylation was monitored using a modification of a protocol by Visser Smit et al. Corneal epithelial cells (primary, hTCEpi, or hTCEpi [-c-Cbl]) were serum starved for 2 hours, treated with 100 ng/mL EGF in K-SFM for 10 minutes, and harvested in a chilled EGFR-UB lysis buffer (0.5% Triton x-100/50 mM Tris pH 7.5/150 mM NaCl/1 mM EDTA/1 mM sodium orthovanadate/10 mM sodium fluoride) supplemented with 2 mM phenylmethyl sulfonyl fluoride (PMSF) (Calbiochem, Billerica, MA, USA)/16 μM G5 Ubiquitin isopeptidase inhibitor I (Santa Cruz Biotechnology).

    Techniques: Migration, Incubation

    Knockdown of c-Cbl reduces EGF-dependent ubiquitylation of the EGFR in corneal epithelial cells. Stable hTCEpi cells lines encoding for tetracycline-regulatable c-Cbl-specific shRNA and DsRed (hTCEpi[-c-Cbl]) were generated as described in Materials and

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Antagonizing c-Cbl Enhances EGFR-Dependent Corneal Epithelial Homeostasis

    doi: 10.1167/iovs.14-14133

    Figure Lengend Snippet: Knockdown of c-Cbl reduces EGF-dependent ubiquitylation of the EGFR in corneal epithelial cells. Stable hTCEpi cells lines encoding for tetracycline-regulatable c-Cbl-specific shRNA and DsRed (hTCEpi[-c-Cbl]) were generated as described in Materials and

    Article Snippet: Epidermal growth factor receptor ubiquitylation was monitored using a modification of a protocol by Visser Smit et al. Corneal epithelial cells (primary, hTCEpi, or hTCEpi [-c-Cbl]) were serum starved for 2 hours, treated with 100 ng/mL EGF in K-SFM for 10 minutes, and harvested in a chilled EGFR-UB lysis buffer (0.5% Triton x-100/50 mM Tris pH 7.5/150 mM NaCl/1 mM EDTA/1 mM sodium orthovanadate/10 mM sodium fluoride) supplemented with 2 mM phenylmethyl sulfonyl fluoride (PMSF) (Calbiochem, Billerica, MA, USA)/16 μM G5 Ubiquitin isopeptidase inhibitor I (Santa Cruz Biotechnology).

    Techniques: shRNA, Generated

    The PP1 attenuates EGF-mediated EGFR ubiquitylation and enhances EGFR corneal epithelial wound healing in vitro. A 1.5-mm-diameter wound was generated on the corneal epithelium of a mouse as described in Materials and Methods. Eyes were treated with various

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Antagonizing c-Cbl Enhances EGFR-Dependent Corneal Epithelial Homeostasis

    doi: 10.1167/iovs.14-14133

    Figure Lengend Snippet: The PP1 attenuates EGF-mediated EGFR ubiquitylation and enhances EGFR corneal epithelial wound healing in vitro. A 1.5-mm-diameter wound was generated on the corneal epithelium of a mouse as described in Materials and Methods. Eyes were treated with various

    Article Snippet: Epidermal growth factor receptor ubiquitylation was monitored using a modification of a protocol by Visser Smit et al. Corneal epithelial cells (primary, hTCEpi, or hTCEpi [-c-Cbl]) were serum starved for 2 hours, treated with 100 ng/mL EGF in K-SFM for 10 minutes, and harvested in a chilled EGFR-UB lysis buffer (0.5% Triton x-100/50 mM Tris pH 7.5/150 mM NaCl/1 mM EDTA/1 mM sodium orthovanadate/10 mM sodium fluoride) supplemented with 2 mM phenylmethyl sulfonyl fluoride (PMSF) (Calbiochem, Billerica, MA, USA)/16 μM G5 Ubiquitin isopeptidase inhibitor I (Santa Cruz Biotechnology).

    Techniques: In Vitro, Generated