phosphorylated kinase antibody array  (R&D Systems)

Journal: Hereditas

Article Title: Regulatory role of ezrin in esophageal cancer progression via the PI3K-AKT signaling pathway

doi: 10.1186/s41065-025-00554-w

Figure Lengend Snippet: Phosphorylated kinase antibody microarray targets

Article Snippet: The phosphorylated kinase antibody array (Catalog No. ARY003C) was obtained from R&D Systems Inc.

Techniques: Microarray

Journal: Nature Communications

Article Title: Cathepsin D deficiency in mammary epithelium transiently stalls breast cancer by interference with mTORC1 signaling

doi: 10.1038/s41467-020-18935-2

Figure Lengend Snippet: a Phospho-kinase antibody arrays with lysates from Ctsd +/+ and Ctsd − /− PyMT cells cultured for 7–10 days (1% FCS) or for ≥8 weeks (1% FCS LT) in 1% FCS medium. Changes in phosphorylation are plotted as log2 fold change (FC) for indicated comparisons ( n = 2 independent experiments). b , c Transcriptome analysis of 1% FCS and 1% FCS LT Ctsd +/+ and Ctsd − /− PyMT cells ( n = 3 independent experiments). b Change in expression of significantly regulated genes of the CREB gene set for indicated comparisons. Red dashed lines, borders for gene up- and downregulation (|log2 FC | > 1). c Clustering analysis of the log2 FC of CREB genes with significant upregulation in Ctsd − /− 1% FCS LT versus 1% FCS PyMT cells for indicated comparisons shown as heatmap. Source data are provided as a Source Data file.

Article Snippet: Lysates from 1% FCS and 1% FCS LT Ctsd +/+ and Ctsd −/− cells were prepared and incubated with membranes spotted with antibodies specific for kinase phosphorylation sites (R&D, ARY003B) following the manufacturer’s instructions.

Techniques: Cell Culture, Phospho-proteomics, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Membranous NOX5-derived ROS oxidizes and activates local Src to promote malignancy of tumor cells

doi: 10.1038/s41392-020-0193-z

Figure Lengend Snippet: NOX5 positively regulates Src activity. a Stable overexpression of NOX5 in the indicated ESCC cell lines tested by immunoblotting. GAPDH was used as a loading control. b Total protein lysates from the vector control or NOX5-overexpressing KYSE30 (upper panel) or KYSE410 (lower panel) cells were analyzed using antibody array against 43 kinase phosphorylation sites. c Silencing NOX5 in two specific short hairpin (sh) RNA-transduced stable ESCC cell lines examined by immunoblotting. GAPDH was used as a loading control. d vector control or NOX5-overexpressing KYSE30 (right panel) or KYSE410 (left panel) cells were pretreated with 10 μM NADPH oxidase inhibitor-DPI or control solvent for 90 min, respectively, or shRNA vector or NOX5 shRNA-1, shRNA-2, were cultured under normoxic and hypoxic conditions for 24 h. The Src activity was assayed by Src activation quantitative ELISA assay. e vector control or NOX5-overexpressing KYSE30 or KYSE410 cells were treated with ROS scavenger NAC (2 mM, pretreated with 90 min) or H 2 O 2 scavenger-PEG-catalase (400 units/ml) or control solvent, respectively, were cultured under normoxic and hypoxic conditions for 24 h. Src activity was assessed using quantitative ELISA assay. *** P < 0.001; two-tailed unpaired Student’s t -test. Error bars represent mean ± SD of five independent experiments. f NOX5 expression was associated with pSrc (Tyr 419 ), expression in 92 primary human ESCC specimens (cohort I). Two representative specimens with low and high levels of NOX5 were shown. Magnification, ×10 as indicated. Percentages of specimens showing low or high NOX5 expression relative to the level of pSrc. Statistical differences were evaluated using the chi-square test

Article Snippet: For the phospho-kinase activation study, antibody arrays against 43 kinase phosphorylation sites (R&D systems; catalog# ARY003) were used according the manufacturer’s instruction.

Techniques: Activity Assay, Over Expression, Western Blot, Control, Plasmid Preparation, Ab Array, Phospho-proteomics, Solvent, shRNA, Cell Culture, Activation Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Membranous NOX5-derived ROS oxidizes and activates local Src to promote malignancy of tumor cells

doi: 10.1038/s41392-020-0193-z

Figure Lengend Snippet: Hypoxia induces cell membranous interaction between Pyk2 and NOX5. a KYSE30 and KYSE410 cells were cultured under normoxic or hypoxic condition for 1 h. Cell membrane lysates were immunoprecipitated with Pyk2 antibody. Immunocomplexes were then immunoblotted using NOX5 and Pyk2 antibodies. The efficacy of membrane protein extraction was examined using immunoblotting to detect the expression of α1-ATPase (membrane biomarker) in cell membrane lysis. b KYSE30 and KYSE410 cells were cultured under normoxic or hypoxic condition for 1 h. Intracellular Ca 2+ level was evaluated using Calcium detection assay kit. c KYSE30 and KYSE410 cells were pretreated with 10 μM Ca 2+ chelator-BAPTA-AM for 30 min and then exposed to normoxic or hypoxic condition for 1 h. The Pyk2 (Tyr 402 ) phosphorylation was assayed by Pyk2 activation ELISA assay. d , e KYSE30 control, NOX5, or Pyk2-ovexexpressing cells were pretreated with 10 μM Ca 2+ chelator-BAPTA-AM for 30 min or control solvent ( d ), or transfected with control vector or Pyk2 Y402F plasmid ( e ), were cultured normoxic or hypoxic condition for 1 h. Cell membrane lysates were immunoprecipitated with Pyk2 antibody. Immunocomplexes were then immunoblotted using NOX5 and Pyk2 antibodies. The efficacy of membrane protein extraction was evaluated using immunoblotting to assess the expression of α1-ATPase (membrane biomarker) in cell membrane lysis. f The KYSE30 and KYSE410 control shRNA or NOX5 shRNA cells were exposed to hypoxia for 1 h. Cell membrane lysates were immunoprecipitated with Pyk2 antibody. Then, the Pyk2 complex-produced H 2 O 2 was examined using an Amplex red hydrogen peroxide assay kit. g KYSE30 control or NOX5-overexpressing cells pretreated with or without 10 μM Ca 2+ chelator-BAPTA-AM, or transfected with control vector or Pyk2 Y402F plasmid, were exposed to normoxic or hypoxic condition for 1 h. Cell membrane lysates were immunoprecipitated with Pyk2 antibody. Then, the Pyk2 complex-produced H 2 O 2 was assayed using an Amplex red hydrogen peroxide assay kit. *** P < 0.001; two-tailed unpaired Student’s t -test. Error bars represent mean ± SD of five independent experiments

Article Snippet: For the phospho-kinase activation study, antibody arrays against 43 kinase phosphorylation sites (R&D systems; catalog# ARY003) were used according the manufacturer’s instruction.

Techniques: Cell Culture, Membrane, Immunoprecipitation, Protein Extraction, Western Blot, Expressing, Biomarker Discovery, Lysis, Detection Assay, Phospho-proteomics, Activation Assay, Enzyme-linked Immunosorbent Assay, Control, Solvent, Transfection, Plasmid Preparation, shRNA, Produced, Amplex Red Hydrogen Peroxide Assay, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Membranous NOX5-derived ROS oxidizes and activates local Src to promote malignancy of tumor cells

doi: 10.1038/s41392-020-0193-z

Figure Lengend Snippet: Pyk2 recruits c-Abl to enhance NOX5 activity in Pyk2/NOX5 complex. a KYSE30 or KYSE410 cells were transfected with vector, Flag-Pyk2 wild type (wt), or Flag- Pyk2 Y881F mutant plasmid and then cultured under normoxia or hypoxia for 1 h. Cell membrane lysates were immunoprecipitated with c-Abl antibody. Immunocomplexes were then immunoblotted using NOX5 and c-Abl antibodies ( a ). Membranous Src was immunoprecipitated with an anti-Src antibody. Oxidized Src levels were measured using a modified OxyBlot protein detection kit ( a ). The efficacy of membrane protein extraction was evaluated using immunoblotting to detect the expression of α1-ATPase (membrane biomarker) ( a ) in cell membrane lysis. Cell membrane lysates were immunoprecipitated with Pyk2 antibody. b–d HeLa cells were co-transfected HA-c-Abl wt or kinase-dead (KD) K290R mutant with Flag-tagged NOX5 wt, Y476/478F, Y487F, or Y519F plasmid, respectively. Cell lysates were immunoprecipitated with the antibody against Flag. Cell membrane lysates were then immunoblotted using antibodies against flag and phosphotyrosine. Membranous Src was immunoprecipitated with an anti-Src antibody. Oxidized Src levels were assayed using a modified OxyBlot protein detection kit ( b ). The transfection efficacy was measured using immunoblotting ( c ). The Pyk2 complex-derived H 2 O 2 was tested using an Amplex red hydrogen peroxide assay kit ( d ). e , f The indicated ESCC cells were transfected with control vector, Flag-NOX5 wt, or Flag-NOX5 Y476/478F (mutant) plasmid, respectively, and then cultured under normoxic or hypoxic condition for 1 h. Oxidized Src levels were measured using a modified OxyBlot protein detection kit ( e ). The phosphorylation of Src Tyr 419 in Pyk2 complex was immunoprecipitated with the antibody against Pyk2 and then immunoblotted using antibodies against Pyk2 and pSrc (Tyr 419 ) ( e ). The efficacy of membrane protein extraction was evaluated using immunoblotting to detect the expression of α1-ATPase (membrane biomarker) ( e ) in cell membrane lysis. The Pyk2 complex-produced H 2 O 2 was assayed using an Amplex red hydrogen peroxide assay kit ( f ). *** P < 0.001; two-tailed unpaired Student’s t -test. Error bars represent mean ± SD of five independent experiments

Article Snippet: For the phospho-kinase activation study, antibody arrays against 43 kinase phosphorylation sites (R&D systems; catalog# ARY003) were used according the manufacturer’s instruction.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Cell Culture, Membrane, Immunoprecipitation, Modification, Protein Extraction, Western Blot, Expressing, Biomarker Discovery, Lysis, Derivative Assay, Amplex Red Hydrogen Peroxide Assay, Control, Phospho-proteomics, Produced, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: Membranous NOX5-derived ROS oxidizes and activates local Src to promote malignancy of tumor cells

doi: 10.1038/s41392-020-0193-z

Figure Lengend Snippet: Proposed Model of Membranous NOX5-derived local ROS oxidizes and activates Src to promote growth and invasion of ESCC cells. Hypoxia stimulated the interaction between NOX5 and Pyk2 on cell membrane via enhancing intracellular Ca 2+ -mediated Pyk2 Tyr 402 phosphorylation. Subsequently, Pyk2 acted as a scaffold via its Tyr 881 site for c-Abl phosphorylating the catalytic domain of NOX5 Tyr 476/478 and then upregulated H 2 O 2 inside the Pyk2/NOX5 complex to oxidize and activate Src

Article Snippet: For the phospho-kinase activation study, antibody arrays against 43 kinase phosphorylation sites (R&D systems; catalog# ARY003) were used according the manufacturer’s instruction.

Techniques: Derivative Assay, Membrane, Phospho-proteomics