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active human egfr kinase domain  (Sino Biological)


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    Sino Biological active human egfr kinase domain
    Active Human Egfr Kinase Domain, supplied by Sino Biological, used in various techniques. Bioz Stars score: 91/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/active+egfr+kinase+domain/EGFR%2C+Active/pmc05130541-123-11-16
    Average 91 stars, based on 16 article reviews
    active human egfr kinase domain - by Bioz Stars, 2026-09
    91/100 stars

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    In Vitro:

    Article Title: The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling
    Article Snippet: Expression of genes of interest was analyzed by quantitative PCR relative to GAPDH using the ΔΔC T method using SYBR Green PCR Supermix (Bio-Rad) and the following primers: mouse NOX2 (forward, GAA TCA GCC TTA GTG TCA CAG G; reverse, ATT CCG GTA TGC GTC CAG C); human DUOX1 (forward, AAC AGA ACA TTG CGA TGT ATG AG; reverse, AGA ATG GAC GGT ATC CTG GA); NOX2 (forward, AAT CAT CCA TGC CAC CAT TT; reverse, TCA AAA TCT GCT GTC CTT CC); Src (forward, GGG TGA TGT TTG ACC TTC AG; reverse, TAG GCA CTC TTT TCC CTC CT) and normalized to GAPDH. .. In Vitro Tyrosine Kinase Assays Tyrosine kinase activity of recombinant active EGFR kinase domain (Signal Chem; sequence 695-end) or active EGFR C797S variant (Signal Chem) was analyzed using the ADP-Glo (Promega) assay kit according to the manufacturer's protocol with the exception of excluding DTT from in the kinase reaction buffers. .. EGFR (∼50 ng) was pretreated at room temperature with hydrogen peroxide (H 2 O 2 ; Thermo) and oxidized glutathione (GSSG; Sigma) for 15 min before initiating catalysis with substrate (poly[4Glu:Tyr]; Sigma) and ATP (Promega) for 60 min at room temperature within the linear kinetic range where changes in percent tyrosine kinase activity reflect difference in initial catalytic velocities.

    Activity Assay:

    Article Title: The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling
    Article Snippet: Expression of genes of interest was analyzed by quantitative PCR relative to GAPDH using the ΔΔC T method using SYBR Green PCR Supermix (Bio-Rad) and the following primers: mouse NOX2 (forward, GAA TCA GCC TTA GTG TCA CAG G; reverse, ATT CCG GTA TGC GTC CAG C); human DUOX1 (forward, AAC AGA ACA TTG CGA TGT ATG AG; reverse, AGA ATG GAC GGT ATC CTG GA); NOX2 (forward, AAT CAT CCA TGC CAC CAT TT; reverse, TCA AAA TCT GCT GTC CTT CC); Src (forward, GGG TGA TGT TTG ACC TTC AG; reverse, TAG GCA CTC TTT TCC CTC CT) and normalized to GAPDH. .. In Vitro Tyrosine Kinase Assays Tyrosine kinase activity of recombinant active EGFR kinase domain (Signal Chem; sequence 695-end) or active EGFR C797S variant (Signal Chem) was analyzed using the ADP-Glo (Promega) assay kit according to the manufacturer's protocol with the exception of excluding DTT from in the kinase reaction buffers. .. EGFR (∼50 ng) was pretreated at room temperature with hydrogen peroxide (H 2 O 2 ; Thermo) and oxidized glutathione (GSSG; Sigma) for 15 min before initiating catalysis with substrate (poly[4Glu:Tyr]; Sigma) and ATP (Promega) for 60 min at room temperature within the linear kinetic range where changes in percent tyrosine kinase activity reflect difference in initial catalytic velocities.

    Article Title: The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling
    Article Snippet: Expression of genes of interest was analyzed by quantitative PCR relative to GAPDH using the ΔΔC T method using SYBR Green PCR Supermix (Bio-Rad) and the following primers: mouse NOX2 (forward, GAA TCA GCC TTA GTG TCA CAG G; reverse, ATT CCG GTA TGC GTC CAG C); human DUOX1 (forward, AAC AGA ACA TTG CGA TGT ATG AG; reverse, AGA ATG GAC GGT ATC CTG GA); NOX2 (forward, AAT CAT CCA TGC CAC CAT TT; reverse, TCA AAA TCT GCT GTC CTT CC); Src (forward, GGG TGA TGT TTG ACC TTC AG; reverse, TAG GCA CTC TTT TCC CTC CT) and normalized to GAPDH. .. Tyrosine kinase activity of recombinant active EGFR kinase domain (Signal Chem; sequence 695-end) or active EGFR C797S variant (Signal Chem) was analyzed using the ADP-Glo (Promega) assay kit according to the manufacturer's protocol with the exception of excluding DTT from in the kinase reaction buffers. .. EGFR (∼50 ng) was pretreated at room temperature with hydrogen peroxide (H 2 O 2 ; Thermo) and oxidized glutathione (GSSG; Sigma) for 15 min before initiating catalysis with substrate (poly[4Glu:Tyr]; Sigma) and ATP (Promega) for 60 min at room temperature within the linear kinetic range where changes in percent tyrosine kinase activity reflect difference in initial catalytic velocities.

    Article Title: The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling
    Article Snippet: Expression of genes of interest was analyzed by qPCR relative to GAPDH using the ΔΔCT method using SYBR Green PCR Supermix (BioRad) and the following primers mouse NOX2 (Forward: GAA TCA GCC TTA GTG TCA CAG G; REVERSE ATT CCG GTA TGC GTC CAG C) and human DUOX1 (Forward; AAC AGA ACA TTG CGA TGT ATG AG; Reverse; AGA ATG GAC GGT ATC CTG GA) NOX2 (Forward: AAT CAT CCA TGC CAC CAT TT; Reverse: TCA AAA TCT GCT GTC CTT CC); Src (Forward: GGG TGA TGT TTG ACC TTC AG; Reverse: TAG GCA CTC TTT TCC CTC CT) and normalized to GAPDH. by guest on O ctober 10, 2016 http://w w w .jbc.org/ D ow nloaded from Redox Regulation of EGFR Signaling 11 In Vitro Tyrosine Kinase Assays. .. Tyrosine kinase activity of recombinant active EGFR kinase domain (Signal Chem; sequence 695-end) or active EGFR C797S variant (Signal Chem) was analyzed using the ADP-Glo (Promega) assay kit according to the manufacture’s protocol with the exception of excluding DTT from in the kinase reaction buffers. .. EGFR (~50 ng) was pre-treated at room temperature with hydrogen peroxide (H2O2; Thermo) and oxidized glutathione (GSSG; Sigma) for 15 minutes prior to initiating catalysis with substrate (poly[4Glu:Tyr]; Sigma) and ATP (Promega) for 60 minutes at room temperature within the linear kinetic range where changes in percent tyrosine kinase activity reflect difference in initial catalytic velocities.

    Recombinant:

    Article Title: The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling
    Article Snippet: Expression of genes of interest was analyzed by quantitative PCR relative to GAPDH using the ΔΔC T method using SYBR Green PCR Supermix (Bio-Rad) and the following primers: mouse NOX2 (forward, GAA TCA GCC TTA GTG TCA CAG G; reverse, ATT CCG GTA TGC GTC CAG C); human DUOX1 (forward, AAC AGA ACA TTG CGA TGT ATG AG; reverse, AGA ATG GAC GGT ATC CTG GA); NOX2 (forward, AAT CAT CCA TGC CAC CAT TT; reverse, TCA AAA TCT GCT GTC CTT CC); Src (forward, GGG TGA TGT TTG ACC TTC AG; reverse, TAG GCA CTC TTT TCC CTC CT) and normalized to GAPDH. .. In Vitro Tyrosine Kinase Assays Tyrosine kinase activity of recombinant active EGFR kinase domain (Signal Chem; sequence 695-end) or active EGFR C797S variant (Signal Chem) was analyzed using the ADP-Glo (Promega) assay kit according to the manufacturer's protocol with the exception of excluding DTT from in the kinase reaction buffers. .. EGFR (∼50 ng) was pretreated at room temperature with hydrogen peroxide (H 2 O 2 ; Thermo) and oxidized glutathione (GSSG; Sigma) for 15 min before initiating catalysis with substrate (poly[4Glu:Tyr]; Sigma) and ATP (Promega) for 60 min at room temperature within the linear kinetic range where changes in percent tyrosine kinase activity reflect difference in initial catalytic velocities.

    Article Title: The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling
    Article Snippet: Expression of genes of interest was analyzed by quantitative PCR relative to GAPDH using the ΔΔC T method using SYBR Green PCR Supermix (Bio-Rad) and the following primers: mouse NOX2 (forward, GAA TCA GCC TTA GTG TCA CAG G; reverse, ATT CCG GTA TGC GTC CAG C); human DUOX1 (forward, AAC AGA ACA TTG CGA TGT ATG AG; reverse, AGA ATG GAC GGT ATC CTG GA); NOX2 (forward, AAT CAT CCA TGC CAC CAT TT; reverse, TCA AAA TCT GCT GTC CTT CC); Src (forward, GGG TGA TGT TTG ACC TTC AG; reverse, TAG GCA CTC TTT TCC CTC CT) and normalized to GAPDH. .. Tyrosine kinase activity of recombinant active EGFR kinase domain (Signal Chem; sequence 695-end) or active EGFR C797S variant (Signal Chem) was analyzed using the ADP-Glo (Promega) assay kit according to the manufacturer's protocol with the exception of excluding DTT from in the kinase reaction buffers. .. EGFR (∼50 ng) was pretreated at room temperature with hydrogen peroxide (H 2 O 2 ; Thermo) and oxidized glutathione (GSSG; Sigma) for 15 min before initiating catalysis with substrate (poly[4Glu:Tyr]; Sigma) and ATP (Promega) for 60 min at room temperature within the linear kinetic range where changes in percent tyrosine kinase activity reflect difference in initial catalytic velocities.

    Article Title: The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling
    Article Snippet: Expression of genes of interest was analyzed by qPCR relative to GAPDH using the ΔΔCT method using SYBR Green PCR Supermix (BioRad) and the following primers mouse NOX2 (Forward: GAA TCA GCC TTA GTG TCA CAG G; REVERSE ATT CCG GTA TGC GTC CAG C) and human DUOX1 (Forward; AAC AGA ACA TTG CGA TGT ATG AG; Reverse; AGA ATG GAC GGT ATC CTG GA) NOX2 (Forward: AAT CAT CCA TGC CAC CAT TT; Reverse: TCA AAA TCT GCT GTC CTT CC); Src (Forward: GGG TGA TGT TTG ACC TTC AG; Reverse: TAG GCA CTC TTT TCC CTC CT) and normalized to GAPDH. by guest on O ctober 10, 2016 http://w w w .jbc.org/ D ow nloaded from Redox Regulation of EGFR Signaling 11 In Vitro Tyrosine Kinase Assays. .. Tyrosine kinase activity of recombinant active EGFR kinase domain (Signal Chem; sequence 695-end) or active EGFR C797S variant (Signal Chem) was analyzed using the ADP-Glo (Promega) assay kit according to the manufacture’s protocol with the exception of excluding DTT from in the kinase reaction buffers. .. EGFR (~50 ng) was pre-treated at room temperature with hydrogen peroxide (H2O2; Thermo) and oxidized glutathione (GSSG; Sigma) for 15 minutes prior to initiating catalysis with substrate (poly[4Glu:Tyr]; Sigma) and ATP (Promega) for 60 minutes at room temperature within the linear kinetic range where changes in percent tyrosine kinase activity reflect difference in initial catalytic velocities.

    Sequencing:

    Article Title: The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling
    Article Snippet: Expression of genes of interest was analyzed by quantitative PCR relative to GAPDH using the ΔΔC T method using SYBR Green PCR Supermix (Bio-Rad) and the following primers: mouse NOX2 (forward, GAA TCA GCC TTA GTG TCA CAG G; reverse, ATT CCG GTA TGC GTC CAG C); human DUOX1 (forward, AAC AGA ACA TTG CGA TGT ATG AG; reverse, AGA ATG GAC GGT ATC CTG GA); NOX2 (forward, AAT CAT CCA TGC CAC CAT TT; reverse, TCA AAA TCT GCT GTC CTT CC); Src (forward, GGG TGA TGT TTG ACC TTC AG; reverse, TAG GCA CTC TTT TCC CTC CT) and normalized to GAPDH. .. In Vitro Tyrosine Kinase Assays Tyrosine kinase activity of recombinant active EGFR kinase domain (Signal Chem; sequence 695-end) or active EGFR C797S variant (Signal Chem) was analyzed using the ADP-Glo (Promega) assay kit according to the manufacturer's protocol with the exception of excluding DTT from in the kinase reaction buffers. .. EGFR (∼50 ng) was pretreated at room temperature with hydrogen peroxide (H 2 O 2 ; Thermo) and oxidized glutathione (GSSG; Sigma) for 15 min before initiating catalysis with substrate (poly[4Glu:Tyr]; Sigma) and ATP (Promega) for 60 min at room temperature within the linear kinetic range where changes in percent tyrosine kinase activity reflect difference in initial catalytic velocities.

    Article Title: The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling
    Article Snippet: Expression of genes of interest was analyzed by quantitative PCR relative to GAPDH using the ΔΔC T method using SYBR Green PCR Supermix (Bio-Rad) and the following primers: mouse NOX2 (forward, GAA TCA GCC TTA GTG TCA CAG G; reverse, ATT CCG GTA TGC GTC CAG C); human DUOX1 (forward, AAC AGA ACA TTG CGA TGT ATG AG; reverse, AGA ATG GAC GGT ATC CTG GA); NOX2 (forward, AAT CAT CCA TGC CAC CAT TT; reverse, TCA AAA TCT GCT GTC CTT CC); Src (forward, GGG TGA TGT TTG ACC TTC AG; reverse, TAG GCA CTC TTT TCC CTC CT) and normalized to GAPDH. .. Tyrosine kinase activity of recombinant active EGFR kinase domain (Signal Chem; sequence 695-end) or active EGFR C797S variant (Signal Chem) was analyzed using the ADP-Glo (Promega) assay kit according to the manufacturer's protocol with the exception of excluding DTT from in the kinase reaction buffers. .. EGFR (∼50 ng) was pretreated at room temperature with hydrogen peroxide (H 2 O 2 ; Thermo) and oxidized glutathione (GSSG; Sigma) for 15 min before initiating catalysis with substrate (poly[4Glu:Tyr]; Sigma) and ATP (Promega) for 60 min at room temperature within the linear kinetic range where changes in percent tyrosine kinase activity reflect difference in initial catalytic velocities.

    Article Title: The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling
    Article Snippet: Expression of genes of interest was analyzed by qPCR relative to GAPDH using the ΔΔCT method using SYBR Green PCR Supermix (BioRad) and the following primers mouse NOX2 (Forward: GAA TCA GCC TTA GTG TCA CAG G; REVERSE ATT CCG GTA TGC GTC CAG C) and human DUOX1 (Forward; AAC AGA ACA TTG CGA TGT ATG AG; Reverse; AGA ATG GAC GGT ATC CTG GA) NOX2 (Forward: AAT CAT CCA TGC CAC CAT TT; Reverse: TCA AAA TCT GCT GTC CTT CC); Src (Forward: GGG TGA TGT TTG ACC TTC AG; Reverse: TAG GCA CTC TTT TCC CTC CT) and normalized to GAPDH. by guest on O ctober 10, 2016 http://w w w .jbc.org/ D ow nloaded from Redox Regulation of EGFR Signaling 11 In Vitro Tyrosine Kinase Assays. .. Tyrosine kinase activity of recombinant active EGFR kinase domain (Signal Chem; sequence 695-end) or active EGFR C797S variant (Signal Chem) was analyzed using the ADP-Glo (Promega) assay kit according to the manufacture’s protocol with the exception of excluding DTT from in the kinase reaction buffers. .. EGFR (~50 ng) was pre-treated at room temperature with hydrogen peroxide (H2O2; Thermo) and oxidized glutathione (GSSG; Sigma) for 15 minutes prior to initiating catalysis with substrate (poly[4Glu:Tyr]; Sigma) and ATP (Promega) for 60 minutes at room temperature within the linear kinetic range where changes in percent tyrosine kinase activity reflect difference in initial catalytic velocities.

    Variant Assay:

    Article Title: The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling
    Article Snippet: Expression of genes of interest was analyzed by quantitative PCR relative to GAPDH using the ΔΔC T method using SYBR Green PCR Supermix (Bio-Rad) and the following primers: mouse NOX2 (forward, GAA TCA GCC TTA GTG TCA CAG G; reverse, ATT CCG GTA TGC GTC CAG C); human DUOX1 (forward, AAC AGA ACA TTG CGA TGT ATG AG; reverse, AGA ATG GAC GGT ATC CTG GA); NOX2 (forward, AAT CAT CCA TGC CAC CAT TT; reverse, TCA AAA TCT GCT GTC CTT CC); Src (forward, GGG TGA TGT TTG ACC TTC AG; reverse, TAG GCA CTC TTT TCC CTC CT) and normalized to GAPDH. .. In Vitro Tyrosine Kinase Assays Tyrosine kinase activity of recombinant active EGFR kinase domain (Signal Chem; sequence 695-end) or active EGFR C797S variant (Signal Chem) was analyzed using the ADP-Glo (Promega) assay kit according to the manufacturer's protocol with the exception of excluding DTT from in the kinase reaction buffers. .. EGFR (∼50 ng) was pretreated at room temperature with hydrogen peroxide (H 2 O 2 ; Thermo) and oxidized glutathione (GSSG; Sigma) for 15 min before initiating catalysis with substrate (poly[4Glu:Tyr]; Sigma) and ATP (Promega) for 60 min at room temperature within the linear kinetic range where changes in percent tyrosine kinase activity reflect difference in initial catalytic velocities.

    Article Title: The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling
    Article Snippet: Expression of genes of interest was analyzed by quantitative PCR relative to GAPDH using the ΔΔC T method using SYBR Green PCR Supermix (Bio-Rad) and the following primers: mouse NOX2 (forward, GAA TCA GCC TTA GTG TCA CAG G; reverse, ATT CCG GTA TGC GTC CAG C); human DUOX1 (forward, AAC AGA ACA TTG CGA TGT ATG AG; reverse, AGA ATG GAC GGT ATC CTG GA); NOX2 (forward, AAT CAT CCA TGC CAC CAT TT; reverse, TCA AAA TCT GCT GTC CTT CC); Src (forward, GGG TGA TGT TTG ACC TTC AG; reverse, TAG GCA CTC TTT TCC CTC CT) and normalized to GAPDH. .. Tyrosine kinase activity of recombinant active EGFR kinase domain (Signal Chem; sequence 695-end) or active EGFR C797S variant (Signal Chem) was analyzed using the ADP-Glo (Promega) assay kit according to the manufacturer's protocol with the exception of excluding DTT from in the kinase reaction buffers. .. EGFR (∼50 ng) was pretreated at room temperature with hydrogen peroxide (H 2 O 2 ; Thermo) and oxidized glutathione (GSSG; Sigma) for 15 min before initiating catalysis with substrate (poly[4Glu:Tyr]; Sigma) and ATP (Promega) for 60 min at room temperature within the linear kinetic range where changes in percent tyrosine kinase activity reflect difference in initial catalytic velocities.

    Article Title: The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling
    Article Snippet: Expression of genes of interest was analyzed by qPCR relative to GAPDH using the ΔΔCT method using SYBR Green PCR Supermix (BioRad) and the following primers mouse NOX2 (Forward: GAA TCA GCC TTA GTG TCA CAG G; REVERSE ATT CCG GTA TGC GTC CAG C) and human DUOX1 (Forward; AAC AGA ACA TTG CGA TGT ATG AG; Reverse; AGA ATG GAC GGT ATC CTG GA) NOX2 (Forward: AAT CAT CCA TGC CAC CAT TT; Reverse: TCA AAA TCT GCT GTC CTT CC); Src (Forward: GGG TGA TGT TTG ACC TTC AG; Reverse: TAG GCA CTC TTT TCC CTC CT) and normalized to GAPDH. by guest on O ctober 10, 2016 http://w w w .jbc.org/ D ow nloaded from Redox Regulation of EGFR Signaling 11 In Vitro Tyrosine Kinase Assays. .. Tyrosine kinase activity of recombinant active EGFR kinase domain (Signal Chem; sequence 695-end) or active EGFR C797S variant (Signal Chem) was analyzed using the ADP-Glo (Promega) assay kit according to the manufacture’s protocol with the exception of excluding DTT from in the kinase reaction buffers. .. EGFR (~50 ng) was pre-treated at room temperature with hydrogen peroxide (H2O2; Thermo) and oxidized glutathione (GSSG; Sigma) for 15 minutes prior to initiating catalysis with substrate (poly[4Glu:Tyr]; Sigma) and ATP (Promega) for 60 minutes at room temperature within the linear kinetic range where changes in percent tyrosine kinase activity reflect difference in initial catalytic velocities.



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    EGFR interacts and phosphorylates HSP70. ( A ) and ( B ) HSP70–EGFR interaction in HCC827 cells. The HSP70–EGFR complex was immunoprecipitated from HCC827 cell lysates using antibodies against HSP70 (panel A) or EGFR (panel B), respectively. The immunoprecipitated HSP70–EGFR complex was analyzed by western blot. ( C ) In vitro EGFR kinase assay detecting Y41 phosphorylation of HSP70. Purified EGFR kinase domain protein was used as the enzyme and purified Flag-tagged HSP70 (WT or Y41F) was used as the protein substrate. HSP70 Y41 phosphorylation was evaluated by western blot using the antibody against Y41 phosphorylated HSP70. ( D ) Enhancement of HSP70 phosphorylation by EGF. HCC827 cells expressing Flag-tagged WT or Y41F HSP70 were untreated or treated with EGF (100 ng/ml, 1 h). The phosphorylation of Flag-tagged HSP70 (WT or Y41F) was analyzed by Co-IP using anti-Flag-tag antibody and western blot using the antibody against Y41 phosphorylated HSP70. ( E ) Inhibition of HSP70 phosphorylation by EGFR–TKIs. HCC827 cells were treated with EGFR–TKIs osimertinib, erlotinib and gefitinib. The status of EGFR phosphorylation, EGFR level, HSP70 phosphorylation and HSP70 level were analyzed by western blot. GADPH was used as the loading control for western blot.

    Journal: Nucleic Acids Research

    Article Title: EGFR-mediated HSP70 phosphorylation facilitates PCNA association with chromatin and DNA replication

    doi: 10.1093/nar/gkae938

    Figure Lengend Snippet: EGFR interacts and phosphorylates HSP70. ( A ) and ( B ) HSP70–EGFR interaction in HCC827 cells. The HSP70–EGFR complex was immunoprecipitated from HCC827 cell lysates using antibodies against HSP70 (panel A) or EGFR (panel B), respectively. The immunoprecipitated HSP70–EGFR complex was analyzed by western blot. ( C ) In vitro EGFR kinase assay detecting Y41 phosphorylation of HSP70. Purified EGFR kinase domain protein was used as the enzyme and purified Flag-tagged HSP70 (WT or Y41F) was used as the protein substrate. HSP70 Y41 phosphorylation was evaluated by western blot using the antibody against Y41 phosphorylated HSP70. ( D ) Enhancement of HSP70 phosphorylation by EGF. HCC827 cells expressing Flag-tagged WT or Y41F HSP70 were untreated or treated with EGF (100 ng/ml, 1 h). The phosphorylation of Flag-tagged HSP70 (WT or Y41F) was analyzed by Co-IP using anti-Flag-tag antibody and western blot using the antibody against Y41 phosphorylated HSP70. ( E ) Inhibition of HSP70 phosphorylation by EGFR–TKIs. HCC827 cells were treated with EGFR–TKIs osimertinib, erlotinib and gefitinib. The status of EGFR phosphorylation, EGFR level, HSP70 phosphorylation and HSP70 level were analyzed by western blot. GADPH was used as the loading control for western blot.

    Article Snippet: Active EGFR kinase domain ( N -terminal GST-tagged human EGFR kinase domain, amino acids 696-end) was purchased from Millipore Sigma (Catalog No.: 14–531).

    Techniques: Immunoprecipitation, Western Blot, In Vitro, Kinase Assay, Phospho-proteomics, Purification, Expressing, Co-Immunoprecipitation Assay, FLAG-tag, Inhibition, Control

    ACR and related thiol-reactive electrophiles inhibit allergen-induced activation of EGFR, ERK1/2, and STAT3. A and B: HBE1 cells were pretreated with ACR (30 min) prior to stimulation with HDM or ATP for 15 min and pEGFR (Y1068), pERK1/2, or pSTAT3, as well as their unphosphorylated forms, were analyzed by Western blot. A: representative Western blots. B: densitometry analysis of pEGFR (Y1068), pERK1/2, or pSTAT3, normalized to unphosphorylated proteins, from 3 experiments in duplicate. *P < 0.05. C and D: HBE1 cells were pretreated with ACR, CUR, CINN, or SFN (30 min) prior to stimulation with ATP for 15 min and pEGFR (Y1068), pERK1/2, or pSTAT3 was determined by Western blot. C: representative Western blots. D: densitometry analysis of pEGFR (Y1068), pERK1/2, or pSTAT3. *P < 0.05.

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

    Article Title: Acrolein and thiol-reactive electrophiles suppress allergen-induced innate airway epithelial responses by inhibition of DUOX1 and EGFR

    doi: 10.1152/ajplung.00276.2016

    Figure Lengend Snippet: ACR and related thiol-reactive electrophiles inhibit allergen-induced activation of EGFR, ERK1/2, and STAT3. A and B: HBE1 cells were pretreated with ACR (30 min) prior to stimulation with HDM or ATP for 15 min and pEGFR (Y1068), pERK1/2, or pSTAT3, as well as their unphosphorylated forms, were analyzed by Western blot. A: representative Western blots. B: densitometry analysis of pEGFR (Y1068), pERK1/2, or pSTAT3, normalized to unphosphorylated proteins, from 3 experiments in duplicate. *P < 0.05. C and D: HBE1 cells were pretreated with ACR, CUR, CINN, or SFN (30 min) prior to stimulation with ATP for 15 min and pEGFR (Y1068), pERK1/2, or pSTAT3 was determined by Western blot. C: representative Western blots. D: densitometry analysis of pEGFR (Y1068), pERK1/2, or pSTAT3. *P < 0.05.

    Article Snippet: Recombinant active human EGFR kinase domain (SignalChem; sequence 695-end) was analyzed using the ADP-Glo assay (Promega) according to the protocols provided by the manufacturer, except that DTT was excluded from the kinase reaction buffer.

    Techniques: Activation Assay, Western Blot

    ACR directly inhibits EGFR activation by modification of C797. A and B: HBE1 cells were exposed to ACR (30 min) and stimulated with EGF (100 ng/ml) for 10 min for analysis of activation of EGFR, STAT3, and ERK 1/2 by Western blot as in Fig. 4. Representative blots (A) and densitometry data from 2 independent experiments (B) are shown. *P < 0.05 compared with untreated control; #P < 0.05 compared with control EGF treatment. C: effect of ACR treatment (30 min) of recombinant EGFR kinase domain on tyrosine kinase activity. D: kinase activity analysis of wild-type and C797S variant form of EGFR. The kinase activity of wild-type enzyme was significantly reduced due to the effect of ACR while C797S variant's activity remained unchanged. *P < 0.05.

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

    Article Title: Acrolein and thiol-reactive electrophiles suppress allergen-induced innate airway epithelial responses by inhibition of DUOX1 and EGFR

    doi: 10.1152/ajplung.00276.2016

    Figure Lengend Snippet: ACR directly inhibits EGFR activation by modification of C797. A and B: HBE1 cells were exposed to ACR (30 min) and stimulated with EGF (100 ng/ml) for 10 min for analysis of activation of EGFR, STAT3, and ERK 1/2 by Western blot as in Fig. 4. Representative blots (A) and densitometry data from 2 independent experiments (B) are shown. *P < 0.05 compared with untreated control; #P < 0.05 compared with control EGF treatment. C: effect of ACR treatment (30 min) of recombinant EGFR kinase domain on tyrosine kinase activity. D: kinase activity analysis of wild-type and C797S variant form of EGFR. The kinase activity of wild-type enzyme was significantly reduced due to the effect of ACR while C797S variant's activity remained unchanged. *P < 0.05.

    Article Snippet: Recombinant active human EGFR kinase domain (SignalChem; sequence 695-end) was analyzed using the ADP-Glo assay (Promega) according to the protocols provided by the manufacturer, except that DTT was excluded from the kinase reaction buffer.

    Techniques: Activation Assay, Modification, Western Blot, Control, Recombinant, Activity Assay, Variant Assay

    ACR exposure induces cysteine modification and carbonylation within DUOX1, EGFR, and Src. A and B: untreated or ACR-treated HBE1 cells (30 min) were lysed in the presence of the thiol-specific reagent EZ-Link Iodoacetyl-LC-Biotin, and avidin-purified proteins as well as whole cell lysates were analyzed by Western blot for DUOX1, EGFR, and Src. A: representative blots. B: densitometry analysis of relative thiol content of DUOX1, EGFR, or Src from 2 experiments in duplicate. *P < 0.05. C: untreated or ACR-treated HBE1 cells (30 min) were derivatized with ARP, and avidin-purified proteins or whole cell lysates (input controls) were evaluated by Western blot for DUOX1, EGFR, or Src. D: mice were exposed to ACR (5 ppm; 4 h) or filtered air, and lung homogenates were derivatized with biotin hydrazide for similar analysis of carbonylated proteins. Representative blots from 2–3 separate experiments are shown.

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

    Article Title: Acrolein and thiol-reactive electrophiles suppress allergen-induced innate airway epithelial responses by inhibition of DUOX1 and EGFR

    doi: 10.1152/ajplung.00276.2016

    Figure Lengend Snippet: ACR exposure induces cysteine modification and carbonylation within DUOX1, EGFR, and Src. A and B: untreated or ACR-treated HBE1 cells (30 min) were lysed in the presence of the thiol-specific reagent EZ-Link Iodoacetyl-LC-Biotin, and avidin-purified proteins as well as whole cell lysates were analyzed by Western blot for DUOX1, EGFR, and Src. A: representative blots. B: densitometry analysis of relative thiol content of DUOX1, EGFR, or Src from 2 experiments in duplicate. *P < 0.05. C: untreated or ACR-treated HBE1 cells (30 min) were derivatized with ARP, and avidin-purified proteins or whole cell lysates (input controls) were evaluated by Western blot for DUOX1, EGFR, or Src. D: mice were exposed to ACR (5 ppm; 4 h) or filtered air, and lung homogenates were derivatized with biotin hydrazide for similar analysis of carbonylated proteins. Representative blots from 2–3 separate experiments are shown.

    Article Snippet: Recombinant active human EGFR kinase domain (SignalChem; sequence 695-end) was analyzed using the ADP-Glo assay (Promega) according to the protocols provided by the manufacturer, except that DTT was excluded from the kinase reaction buffer.

    Techniques: Modification, Avidin-Biotin Assay, Purification, Western Blot