Structured Review

Millipore na3 vo4
Na3 Vo4, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 1362 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/na3 vo4/product/Millipore
Average 92 stars, based on 1362 article reviews
Price from $9.99 to $1999.99
na3 vo4 - by Bioz Stars, 2020-07
92/100 stars

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Protease Inhibitor:

Article Title: Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells
Article Snippet: .. Total protein was extracted from cells via lysing in ice-cold modified RIPA buffer with 1 mM Na3 VO4 , 20 mM β-glycerophosphate, sodium deoxycholate, with 1 mM PMSF, and 1% (v/v) protease inhibitor cocktail solution (Sigma, St. Louis, MO) freshly added. .. Up to 50 µg of protein from cell lysates was separated on 7.5% or 4–12% SDS-polyacrylamide and electrophoretically transferred to nitrocellulose membranes (BioRad, Hercules, CA).

Article Title: The evidence of metabolic-improving effect of metformin in Ay/a mice with genetically-induced melanocortin obesity and the contribution of hypothalamic mechanisms to this effect
Article Snippet: .. To determine the intrahypothalamic leptin and insulin content, the samples of the hypothalamus tissue were homogenized in the ratio 1:10 in the lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.25 M sucrose, 0.5% Triton X-100, 0.5% sodium deoxycholate, 15 mM NaF, 10 mM sodium glycerophosphate, 10 mM sodium pyrophosphate, 1 mM Na3 VO4 , 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.02% NaN3 , and the protease inhibitor cocktail (“Sigma-Aldrich”, USA). .. The obtained homogenate was centrifuged (10 000 g , 5 min), and the concentration of leptin and insulin in the supernatant fraction was measured according to the manufacturer's instructions.

Article Title: Split Renilla Luciferase Protein Fragment-assisted Complementation (SRL-PFAC) to Characterize Hsp90-Cdc37 Complex and Identify Critical Residues in Protein/Protein Interactions *
Article Snippet: .. After culturing for 48 h, cells were washed twice with ice-cold phosphate-buffered saline and collected in lysis buffer (20 mmol/liter Tris (pH 7.5), 1% Nonidet P-40, 150 mmol/liter NaCl, 5 mmol/liter EDTA, 1 mmol/liter Na3 VO4 ) supplemented with a protease inhibitor mixture (Sigma; added at a 1:100 dilution) as described previously ( ). .. Equal amounts of protein were subjected to SDS-PAGE.

Article Title: Interactome analysis reveals ZNF804A, a schizophrenia risk gene, as a novel component of protein translational machinery critical for embryonic neurodevelopment
Article Snippet: .. The cell monolayers were washed three times with ice-cold PBS containing 100 μg ml−1 CHX, and 3–5 × 106 cells were lysed in 0.5 ml buffer containing 5 mM Tris-HCl, pH 7.5, 2.5 mM MgCl2 , 1.5 mM KCl, 100 μg ml−1 CHX, 0.5% Triton X-100, 0.5% sodium deoxycholate, 1 mM phenylmethyl sulfonyl fluoride (PMSF), 1 mM Na3 VO4 , 1 mM NaF and 10 μl ml−1 protease inhibitor mixture P8340 (Sigma). .. The EDTA treatment group was treated with 30 mm EDTA after cell lysis.

Article Title: Cognitive deficits and Alzheimer-like neuropathological impairments during adolescence in a rat model of type 2 diabetes mellitus
Article Snippet: .. Hippocampi were homogenized in buffer containing: NaCl 50 mM, Tris (Sigma-Aldrich) 10 mM, ethylenediamine tetraacetic acid 1 mM, Na3 VO4 0.5 mM, NaF 50 mM, phenylmethyl sulfonylfluoride 1 mM, and a protease-inhibitor cocktail (Sigma-Aldrich; P8340). ..

Article Title: Activation of purinergic receptors induces proliferation and neuronal differentiation in Swiss Webster mouse olfactory epithelium
Article Snippet: .. Olfactory epithelium dissected from adult animals treated as previously described (above) were obtained from 6–9 mice and homogenized by sonication in Tris buffer [10 mM Tris-HCl (pH 7.6) containing 100 mM NaCl, 1 mM EDTA, 2 M activated Na3 VO4 , 50 mM NaF, and a protease inhibitor cocktail (1:1000, Sigma-Aldrich, St. Louis, MO)]. .. The concentration of protein in the homogenate was measured using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL).

Infection:

Article Title: Host-directed kinase inhibitors act as novel therapies against intracellular Staphylococcus aureus
Article Snippet: .. Phosphoproteomics of S. aureus -infected cells After 6 hours of MRSA infection, cell extracts were obtained by lysing the cells with 50 µl of lysis buffer (20 mM HEPES (pH 8.0)) buffer containing 8 M urea, 1 mM Na3 VO4 , 1 mM NaF, 1 mM β-glycerol phosphate and 2.5 mM Na2 H2 P2 O7 ; Sigma-Aldrich) per well. .. After lysis buffer was added to the cells, samples were thoroughly vortexed for 5 min and immediately stored at −80 °C.

Mouse Assay:

Article Title: Activation of purinergic receptors induces proliferation and neuronal differentiation in Swiss Webster mouse olfactory epithelium
Article Snippet: .. Olfactory epithelium dissected from adult animals treated as previously described (above) were obtained from 6–9 mice and homogenized by sonication in Tris buffer [10 mM Tris-HCl (pH 7.6) containing 100 mM NaCl, 1 mM EDTA, 2 M activated Na3 VO4 , 50 mM NaF, and a protease inhibitor cocktail (1:1000, Sigma-Aldrich, St. Louis, MO)]. .. The concentration of protein in the homogenate was measured using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL).

other:

Article Title: Distinct patterns of transcriptional and epigenetic alterations characterize acute and chronic kidney injury
Article Snippet: Formaldehyde, ethanol, NaCl, EDTA, Triton X-100, NP-40, Tris, leupeptin, PMSF, p-nitrophenyl phosphate, NaF, Na3 VO4 , Na2 MoO4 and β-glycerophosphate were from Sigma.

Lysis:

Article Title: Host-directed kinase inhibitors act as novel therapies against intracellular Staphylococcus aureus
Article Snippet: .. Phosphoproteomics of S. aureus -infected cells After 6 hours of MRSA infection, cell extracts were obtained by lysing the cells with 50 µl of lysis buffer (20 mM HEPES (pH 8.0)) buffer containing 8 M urea, 1 mM Na3 VO4 , 1 mM NaF, 1 mM β-glycerol phosphate and 2.5 mM Na2 H2 P2 O7 ; Sigma-Aldrich) per well. .. After lysis buffer was added to the cells, samples were thoroughly vortexed for 5 min and immediately stored at −80 °C.

Article Title: The evidence of metabolic-improving effect of metformin in Ay/a mice with genetically-induced melanocortin obesity and the contribution of hypothalamic mechanisms to this effect
Article Snippet: .. To determine the intrahypothalamic leptin and insulin content, the samples of the hypothalamus tissue were homogenized in the ratio 1:10 in the lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.25 M sucrose, 0.5% Triton X-100, 0.5% sodium deoxycholate, 15 mM NaF, 10 mM sodium glycerophosphate, 10 mM sodium pyrophosphate, 1 mM Na3 VO4 , 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.02% NaN3 , and the protease inhibitor cocktail (“Sigma-Aldrich”, USA). .. The obtained homogenate was centrifuged (10 000 g , 5 min), and the concentration of leptin and insulin in the supernatant fraction was measured according to the manufacturer's instructions.

Article Title: Split Renilla Luciferase Protein Fragment-assisted Complementation (SRL-PFAC) to Characterize Hsp90-Cdc37 Complex and Identify Critical Residues in Protein/Protein Interactions *
Article Snippet: .. After culturing for 48 h, cells were washed twice with ice-cold phosphate-buffered saline and collected in lysis buffer (20 mmol/liter Tris (pH 7.5), 1% Nonidet P-40, 150 mmol/liter NaCl, 5 mmol/liter EDTA, 1 mmol/liter Na3 VO4 ) supplemented with a protease inhibitor mixture (Sigma; added at a 1:100 dilution) as described previously ( ). .. Equal amounts of protein were subjected to SDS-PAGE.

Modification:

Article Title: Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells
Article Snippet: .. Total protein was extracted from cells via lysing in ice-cold modified RIPA buffer with 1 mM Na3 VO4 , 20 mM β-glycerophosphate, sodium deoxycholate, with 1 mM PMSF, and 1% (v/v) protease inhibitor cocktail solution (Sigma, St. Louis, MO) freshly added. .. Up to 50 µg of protein from cell lysates was separated on 7.5% or 4–12% SDS-polyacrylamide and electrophoretically transferred to nitrocellulose membranes (BioRad, Hercules, CA).

Sonication:

Article Title: Activation of purinergic receptors induces proliferation and neuronal differentiation in Swiss Webster mouse olfactory epithelium
Article Snippet: .. Olfactory epithelium dissected from adult animals treated as previously described (above) were obtained from 6–9 mice and homogenized by sonication in Tris buffer [10 mM Tris-HCl (pH 7.6) containing 100 mM NaCl, 1 mM EDTA, 2 M activated Na3 VO4 , 50 mM NaF, and a protease inhibitor cocktail (1:1000, Sigma-Aldrich, St. Louis, MO)]. .. The concentration of protein in the homogenate was measured using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL).

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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 3 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 3 article reviews
    Price from $9.99 to $1999.99
    sodium orthovanadate naova - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    99
    Millipore atm kinase buffer
    Cdk5 regulates CPT-induced the re-entry of cell cycle by postmitotic neurons ( a ) Kinetics of CPT-induced activation of <t>Cdks</t> and <t>ATM.</t> CGNs were treated with 10 μM CPT for the indicated periods of time. Cdk5, ATM, Cdk2 and Cdk6 kinase activities were measured following immunoprecipitation. The lower graph is the quantification of various kinase activities. Data were shown as mean ± SD (n = 3. All Ns represent independent experiments throughout). ( b ) Phosphorylation of ATM by Cdks in vitro . Purified Cdk2, 5, and 6 were incubated with the recombinant GST-ATM4 protein in vitro in the presence of [γ- 32 P]-ATP under manufacturer’s recommended conditions (top panel) or with the standard control substrate Histone H1 (bottom panel). ( c ) The effects of dominant negative (dn) Cdks on CPT-induced γ-H2AX foci formation. CGNs were transfected with constructs for GFP and dnCdks. After 24 hours, cells were treated with 10 μM CPT for 1 hour. γ-H2AX (red) and GFP (green) were detected by immunocytochemistry, and nuclei were labeled with Hoechst (blue). The scale bar represents 5 μM. The average number of foci counted blindly are: GFP control, 0.22; CPT+GFP, 4.81; CPT+dnCdk2, 4.56; CPT+dnCdk5, 3.25, p
    Atm Kinase 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/atm kinase buffer/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    atm kinase buffer - by Bioz Stars, 2020-07
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      Buy from Supplier

    92
    Millipore na3 vo4
    Cdk5 regulates CPT-induced the re-entry of cell cycle by postmitotic neurons ( a ) Kinetics of CPT-induced activation of <t>Cdks</t> and <t>ATM.</t> CGNs were treated with 10 μM CPT for the indicated periods of time. Cdk5, ATM, Cdk2 and Cdk6 kinase activities were measured following immunoprecipitation. The lower graph is the quantification of various kinase activities. Data were shown as mean ± SD (n = 3. All Ns represent independent experiments throughout). ( b ) Phosphorylation of ATM by Cdks in vitro . Purified Cdk2, 5, and 6 were incubated with the recombinant GST-ATM4 protein in vitro in the presence of [γ- 32 P]-ATP under manufacturer’s recommended conditions (top panel) or with the standard control substrate Histone H1 (bottom panel). ( c ) The effects of dominant negative (dn) Cdks on CPT-induced γ-H2AX foci formation. CGNs were transfected with constructs for GFP and dnCdks. After 24 hours, cells were treated with 10 μM CPT for 1 hour. γ-H2AX (red) and GFP (green) were detected by immunocytochemistry, and nuclei were labeled with Hoechst (blue). The scale bar represents 5 μM. The average number of foci counted blindly are: GFP control, 0.22; CPT+GFP, 4.81; CPT+dnCdk2, 4.56; CPT+dnCdk5, 3.25, p
    Na3 Vo4, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 1362 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/na3 vo4/product/Millipore
    Average 92 stars, based on 1362 article reviews
    Price from $9.99 to $1999.99
    na3 vo4 - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

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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

    Cdk5 regulates CPT-induced the re-entry of cell cycle by postmitotic neurons ( a ) Kinetics of CPT-induced activation of Cdks and ATM. CGNs were treated with 10 μM CPT for the indicated periods of time. Cdk5, ATM, Cdk2 and Cdk6 kinase activities were measured following immunoprecipitation. The lower graph is the quantification of various kinase activities. Data were shown as mean ± SD (n = 3. All Ns represent independent experiments throughout). ( b ) Phosphorylation of ATM by Cdks in vitro . Purified Cdk2, 5, and 6 were incubated with the recombinant GST-ATM4 protein in vitro in the presence of [γ- 32 P]-ATP under manufacturer’s recommended conditions (top panel) or with the standard control substrate Histone H1 (bottom panel). ( c ) The effects of dominant negative (dn) Cdks on CPT-induced γ-H2AX foci formation. CGNs were transfected with constructs for GFP and dnCdks. After 24 hours, cells were treated with 10 μM CPT for 1 hour. γ-H2AX (red) and GFP (green) were detected by immunocytochemistry, and nuclei were labeled with Hoechst (blue). The scale bar represents 5 μM. The average number of foci counted blindly are: GFP control, 0.22; CPT+GFP, 4.81; CPT+dnCdk2, 4.56; CPT+dnCdk5, 3.25, p

    Journal: Nature cell biology

    Article Title: Phosphorylation of ATM by Cdk5 mediates DNA damage signaling and regulates neuronal death

    doi: 10.1038/ncb1829

    Figure Lengend Snippet: Cdk5 regulates CPT-induced the re-entry of cell cycle by postmitotic neurons ( a ) Kinetics of CPT-induced activation of Cdks and ATM. CGNs were treated with 10 μM CPT for the indicated periods of time. Cdk5, ATM, Cdk2 and Cdk6 kinase activities were measured following immunoprecipitation. The lower graph is the quantification of various kinase activities. Data were shown as mean ± SD (n = 3. All Ns represent independent experiments throughout). ( b ) Phosphorylation of ATM by Cdks in vitro . Purified Cdk2, 5, and 6 were incubated with the recombinant GST-ATM4 protein in vitro in the presence of [γ- 32 P]-ATP under manufacturer’s recommended conditions (top panel) or with the standard control substrate Histone H1 (bottom panel). ( c ) The effects of dominant negative (dn) Cdks on CPT-induced γ-H2AX foci formation. CGNs were transfected with constructs for GFP and dnCdks. After 24 hours, cells were treated with 10 μM CPT for 1 hour. γ-H2AX (red) and GFP (green) were detected by immunocytochemistry, and nuclei were labeled with Hoechst (blue). The scale bar represents 5 μM. The average number of foci counted blindly are: GFP control, 0.22; CPT+GFP, 4.81; CPT+dnCdk2, 4.56; CPT+dnCdk5, 3.25, p

    Article Snippet: To determine kinase activity of ATM or Cdks in cells, corresponding immunoprecipitates were washed twice with either ATM kinase buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 4 mM MnCl2 , 10% glycerol, 1 mM dithiothreitol, and 100 μM Na3 VO4 ) or Cdk kinase buffer, and incubated in the kinase buffer containing [γ-32 P]-ATP and 1 μg of substrate [PHAS-I for ATM kinase assay (Calbiochem) and histone H1 for Cdk kinase assay (Upstate)] for 30 min at 30°C.

    Techniques: Cycling Probe Technology, Activation Assay, Immunoprecipitation, In Vitro, Purification, Incubation, Recombinant, Dominant Negative Mutation, Transfection, Construct, Immunocytochemistry, Labeling