Western Blot:Article Title: Redox-dependent activation of Src kinase mediates epithelial IL-33 production and signaling during acute airway allergen challenge
Article Snippet: Control studies showed that neither HDM, AZD0530, or AG1478 caused significant loss of viability at the doses used, assessed using CellTiter-Glo Viability Assay (Promega, Madison, WI). .. Following treatments, cells were lysed utilizing Western Solubilization Buffer (WSB, 50 mM HEPES, 250 mM NaCl, 1.5 mM MgCl 2 , 1% Triton-X100, 10% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na 3 VO 4 , 10 mg/ml aprotinin, and 10 mg/ml leupeptin (pH 7.4)) or GeneJet RNA extraction kit lysis buffer (Invitrogen) supplemented with 2% β-mercaptoethanol, for analysis of protein or RNA, respectively. .. Silencing of gene expression in H292 cells or MTEC was carried out when cells were 60–70% confluent using targeted siRNAs and the DharmaFECT reagent (Dharmacon), according to manufacturer protocol.
Article Title: DUOX1 mediates persistent epithelial EGFR activation, mucous cell metaplasia, and airway remodeling during allergic asthma
Article Snippet: .. Cell lysates or lung homogenates were prepared using Western solubilization buffer (50 mM HEPES, 250 mM NaCl, 1.5 mM MgCl 2 , 1% Triton X-100, 10% glycerol, 1 mM EGTA, 1 mM PMSF, 2 mM Na 3 VO 4 , 10 μg/ml aprotinin, 10 μg/ml leupeptin; pH 7.4), and samples containing equal amounts of protein (measured using the BCA protein assay kit; Pierce) were separated on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with antibodies against EGFR (C74B9; 1:1,000), pEGFR Tyr845 (D63B4; 1:500), pEGFR Tyr1068 (D7A5; 1:1,000), and Src (L4A1; 1:1,000) (all from Cell Signaling); p-Src Tyr416 (1:1,000; 07-909; Millipore); or DUOX1 (provided by F. Miot, Free University of Brussels, Brussels, Belgium) ( 62 ). .. Primarily antibodies were probed with rabbit- or mouse-specific secondary antibodies conjugated with HRP (Cell Signaling) and detected by chemiluminescence using SuperSignal West Pico Chemiluminescent Substrate (Pierce).
RNA Extraction:Article Title: Redox-dependent activation of Src kinase mediates epithelial IL-33 production and signaling during acute airway allergen challenge
Article Snippet: Control studies showed that neither HDM, AZD0530, or AG1478 caused significant loss of viability at the doses used, assessed using CellTiter-Glo Viability Assay (Promega, Madison, WI). .. Following treatments, cells were lysed utilizing Western Solubilization Buffer (WSB, 50 mM HEPES, 250 mM NaCl, 1.5 mM MgCl 2 , 1% Triton-X100, 10% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na 3 VO 4 , 10 mg/ml aprotinin, and 10 mg/ml leupeptin (pH 7.4)) or GeneJet RNA extraction kit lysis buffer (Invitrogen) supplemented with 2% β-mercaptoethanol, for analysis of protein or RNA, respectively. .. Silencing of gene expression in H292 cells or MTEC was carried out when cells were 60–70% confluent using targeted siRNAs and the DharmaFECT reagent (Dharmacon), according to manufacturer protocol.
Lysis:Article Title: Redox-dependent activation of Src kinase mediates epithelial IL-33 production and signaling during acute airway allergen challenge
Article Snippet: Control studies showed that neither HDM, AZD0530, or AG1478 caused significant loss of viability at the doses used, assessed using CellTiter-Glo Viability Assay (Promega, Madison, WI). .. Following treatments, cells were lysed utilizing Western Solubilization Buffer (WSB, 50 mM HEPES, 250 mM NaCl, 1.5 mM MgCl 2 , 1% Triton-X100, 10% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na 3 VO 4 , 10 mg/ml aprotinin, and 10 mg/ml leupeptin (pH 7.4)) or GeneJet RNA extraction kit lysis buffer (Invitrogen) supplemented with 2% β-mercaptoethanol, for analysis of protein or RNA, respectively. .. Silencing of gene expression in H292 cells or MTEC was carried out when cells were 60–70% confluent using targeted siRNAs and the DharmaFECT reagent (Dharmacon), according to manufacturer protocol.
Analysis:Article Title: Redox-dependent activation of Src kinase mediates epithelial IL-33 production and signaling during acute airway allergen challenge
Article Snippet: Control studies showed that neither HDM, AZD0530, or AG1478 caused significant loss of viability at the doses used, assessed using CellTiter-Glo Viability Assay (Promega, Madison, WI). .. Following treatments, cells were lysed utilizing Western Solubilization Buffer (WSB, 50 mM HEPES, 250 mM NaCl, 1.5 mM MgCl 2 , 1% Triton-X100, 10% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na 3 VO 4 , 10 mg/ml aprotinin, and 10 mg/ml leupeptin (pH 7.4)) or GeneJet RNA extraction kit lysis buffer (Invitrogen) supplemented with 2% β-mercaptoethanol, for analysis of protein or RNA, respectively. .. Silencing of gene expression in H292 cells or MTEC was carried out when cells were 60–70% confluent using targeted siRNAs and the DharmaFECT reagent (Dharmacon), according to manufacturer protocol.
Bicinchoninic Acid Protein Assay:Article Title: DUOX1 mediates persistent epithelial EGFR activation, mucous cell metaplasia, and airway remodeling during allergic asthma
Article Snippet: .. Cell lysates or lung homogenates were prepared using Western solubilization buffer (50 mM HEPES, 250 mM NaCl, 1.5 mM MgCl 2 , 1% Triton X-100, 10% glycerol, 1 mM EGTA, 1 mM PMSF, 2 mM Na 3 VO 4 , 10 μg/ml aprotinin, 10 μg/ml leupeptin; pH 7.4), and samples containing equal amounts of protein (measured using the BCA protein assay kit; Pierce) were separated on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with antibodies against EGFR (C74B9; 1:1,000), pEGFR Tyr845 (D63B4; 1:500), pEGFR Tyr1068 (D7A5; 1:1,000), and Src (L4A1; 1:1,000) (all from Cell Signaling); p-Src Tyr416 (1:1,000; 07-909; Millipore); or DUOX1 (provided by F. Miot, Free University of Brussels, Brussels, Belgium) ( 62 ). .. Primarily antibodies were probed with rabbit- or mouse-specific secondary antibodies conjugated with HRP (Cell Signaling) and detected by chemiluminescence using SuperSignal West Pico Chemiluminescent Substrate (Pierce).
SDS Page:Article Title: DUOX1 mediates persistent epithelial EGFR activation, mucous cell metaplasia, and airway remodeling during allergic asthma
Article Snippet: .. Cell lysates or lung homogenates were prepared using Western solubilization buffer (50 mM HEPES, 250 mM NaCl, 1.5 mM MgCl 2 , 1% Triton X-100, 10% glycerol, 1 mM EGTA, 1 mM PMSF, 2 mM Na 3 VO 4 , 10 μg/ml aprotinin, 10 μg/ml leupeptin; pH 7.4), and samples containing equal amounts of protein (measured using the BCA protein assay kit; Pierce) were separated on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with antibodies against EGFR (C74B9; 1:1,000), pEGFR Tyr845 (D63B4; 1:500), pEGFR Tyr1068 (D7A5; 1:1,000), and Src (L4A1; 1:1,000) (all from Cell Signaling); p-Src Tyr416 (1:1,000; 07-909; Millipore); or DUOX1 (provided by F. Miot, Free University of Brussels, Brussels, Belgium) ( 62 ). .. Primarily antibodies were probed with rabbit- or mouse-specific secondary antibodies conjugated with HRP (Cell Signaling) and detected by chemiluminescence using SuperSignal West Pico Chemiluminescent Substrate (Pierce).
|
Thermo Fisher is a verified supplier 
Thermo Fisher manufactures this product 