human p22 phox cyba  (OriGene)


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    Structured Review

    OriGene human p22 phox cyba
    (A-C) Mouse constructs encoding EROS and gp91 <t>phox</t> were co-transfected into NIH3T3 (A) , COS-7 (B) and HEK293T (C) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. (D-F) gp91 phox and <t>p22</t> phox expression in HEK293T cells following transfection with the indicated human constructs. (G) Analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in presence or absence of EROS and treatment with 10μg/mL cycloheximide. Actin and vinculin were used as loading control. (H) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector and treated with 10μg/mL cycloheximide. (I-J) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox or both in differentiated PL985 knock-out for p22 phox (I) or EROS (J) . n: representative of at least 3 independent experiments. See also Figure S1.
    Human P22 Phox Cyba, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "EROS-mediated control of NOX2 and P2X7 biosynthesis"

    Article Title: EROS-mediated control of NOX2 and P2X7 biosynthesis

    Journal: bioRxiv

    doi: 10.1101/2021.09.14.460103

    (A-C) Mouse constructs encoding EROS and gp91 phox were co-transfected into NIH3T3 (A) , COS-7 (B) and HEK293T (C) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. (D-F) gp91 phox and p22 phox expression in HEK293T cells following transfection with the indicated human constructs. (G) Analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in presence or absence of EROS and treatment with 10μg/mL cycloheximide. Actin and vinculin were used as loading control. (H) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector and treated with 10μg/mL cycloheximide. (I-J) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox or both in differentiated PL985 knock-out for p22 phox (I) or EROS (J) . n: representative of at least 3 independent experiments. See also Figure S1.
    Figure Legend Snippet: (A-C) Mouse constructs encoding EROS and gp91 phox were co-transfected into NIH3T3 (A) , COS-7 (B) and HEK293T (C) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. (D-F) gp91 phox and p22 phox expression in HEK293T cells following transfection with the indicated human constructs. (G) Analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in presence or absence of EROS and treatment with 10μg/mL cycloheximide. Actin and vinculin were used as loading control. (H) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector and treated with 10μg/mL cycloheximide. (I-J) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox or both in differentiated PL985 knock-out for p22 phox (I) or EROS (J) . n: representative of at least 3 independent experiments. See also Figure S1.

    Techniques Used: Construct, Transfection, Expressing, Western Blot, Plasmid Preparation, Transduction, Knock-Out

    (A-D) Immunoprecipitation (IP) and size exclusion chromatography (SEC) analysis of protein complexes associated with EROS. (A) IP of EROS in HEK293-F cells expressing StrepII-FLAG-tagged EROS, gp91 phox -GFP and p22 phox with western blot for gp91 phox . Lysates treated with Peptide N-glycosidase F (PNGaseF) or Endoglycosidase H (EndoH) served as reference; PG: partially glycosylated; NG: non-glycosylated; RT: run through (B) SEC profile of EROS-IP eluate indicating protein (280nm) and heme (414nm) content. (C) Immunoblot analysis of gp91 phox -GFP, EROS-FLAG and endogenous p22 phox in SEC fraction 9-14 and 15-18. (D) SEC profile of EROS eluate from HEK293-F cells expressing EROS-FLAG, gp91 phox and p22 phox constructs and treated with heme biosynthesis inhibitor succinyl acetone (10µg/ml). (E) IP of StrepII-FLAG-tagged EROS in HEK293-F treated with succinyl acetone. (F) Interaction between gp91 phox and EROS assessed through luminescence production in live HEK293T cells expressing the indicated plasmids fused with the large (LgBIT) or small (SmBIT) fragment of the NanoLuc luciferase (see methods). Halo Tag (HT)-SmBIT is the negative control; RLU: Relative Luminescence Unit. ( G) Yeast growth phenotypes obtained with the specified selective media using gp91 phox bait plasmid and EROS prey plasmid. DBD: DNA binding domain of Gal4; AD: Activation domain of Gal4 (see methods). (H) EROS localisation in HEK293 cells transfected with EROS construct (top panel; 3D stack) or EROS and Lap2-GFP constructs (bottom panel; single plane), fixed, permeabilised and labelled with anti-EROS and anti-calnexin antibodies. Scale bar= 5μm. n= representative of at least 3 independent experiments. See also Figure S2.
    Figure Legend Snippet: (A-D) Immunoprecipitation (IP) and size exclusion chromatography (SEC) analysis of protein complexes associated with EROS. (A) IP of EROS in HEK293-F cells expressing StrepII-FLAG-tagged EROS, gp91 phox -GFP and p22 phox with western blot for gp91 phox . Lysates treated with Peptide N-glycosidase F (PNGaseF) or Endoglycosidase H (EndoH) served as reference; PG: partially glycosylated; NG: non-glycosylated; RT: run through (B) SEC profile of EROS-IP eluate indicating protein (280nm) and heme (414nm) content. (C) Immunoblot analysis of gp91 phox -GFP, EROS-FLAG and endogenous p22 phox in SEC fraction 9-14 and 15-18. (D) SEC profile of EROS eluate from HEK293-F cells expressing EROS-FLAG, gp91 phox and p22 phox constructs and treated with heme biosynthesis inhibitor succinyl acetone (10µg/ml). (E) IP of StrepII-FLAG-tagged EROS in HEK293-F treated with succinyl acetone. (F) Interaction between gp91 phox and EROS assessed through luminescence production in live HEK293T cells expressing the indicated plasmids fused with the large (LgBIT) or small (SmBIT) fragment of the NanoLuc luciferase (see methods). Halo Tag (HT)-SmBIT is the negative control; RLU: Relative Luminescence Unit. ( G) Yeast growth phenotypes obtained with the specified selective media using gp91 phox bait plasmid and EROS prey plasmid. DBD: DNA binding domain of Gal4; AD: Activation domain of Gal4 (see methods). (H) EROS localisation in HEK293 cells transfected with EROS construct (top panel; 3D stack) or EROS and Lap2-GFP constructs (bottom panel; single plane), fixed, permeabilised and labelled with anti-EROS and anti-calnexin antibodies. Scale bar= 5μm. n= representative of at least 3 independent experiments. See also Figure S2.

    Techniques Used: Immunoprecipitation, Size-exclusion Chromatography, Expressing, Western Blot, Construct, Luciferase, Negative Control, Plasmid Preparation, Binding Assay, Activation Assay, Transfection

    human p22 phox sirna  (Thermo Fisher)


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    Structured Review

    Thermo Fisher human p22 phox sirna
    ( A ) Identification of NOX expressed in neuroblastoma (SK-N-BE) and colon carcinoma (Caco-2) cells. Total RNA was extracted with Trizol, reverse transcribed and analyzed by PCR (see ) with specific primers to NOX1, NOX2 and NOX4 (left) or NOX3 and NOX5 (right). The number of cycles was 35. ( B ) Cells were transfected by electroporation with two different (45 and 46) <t>siRNA</t> to <t>p22</t> <t>phox</t> (siRNA p22 phox ) or control, scrambled siRNA (scramble) as described in . 48h after transfection, total proteins were extracted and subjected to immunoblot analysis of p22 phox . The histograms show the mean +/- SEM values relative to scramble obtained by densitometric analysis of p22 phox bands normalized for α-tubulin of three independent experiments. *p< 0.01 vs scramble. ( C ) 24h after transfection cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min. Total proteins were extracted and subjected to immunoblot analysis of DUOX. The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) obtained by densitometric analysis of DUOX bands normalized for α-tubulin of three independent experiments. * p< 0.01 vs Ctr. ( D, E ) 24h after transfection with a mix of the two p22 phox siRNA, cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min, mRNA was extracted and DUOX1 and DUOX2 mRNA levels were analyzed by RT-PCR as described in . The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) of three independent experiments. * p<0.05 vs Ctr; ** p<0.05 vs PDGF stimulated scramble.
    Human P22 Phox Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Reactive Oxygen Species Regulate the Levels of Dual Oxidase (Duox1-2) in Human Neuroblastoma Cells"

    Article Title: Reactive Oxygen Species Regulate the Levels of Dual Oxidase (Duox1-2) in Human Neuroblastoma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034405

    ( A ) Identification of NOX expressed in neuroblastoma (SK-N-BE) and colon carcinoma (Caco-2) cells. Total RNA was extracted with Trizol, reverse transcribed and analyzed by PCR (see ) with specific primers to NOX1, NOX2 and NOX4 (left) or NOX3 and NOX5 (right). The number of cycles was 35. ( B ) Cells were transfected by electroporation with two different (45 and 46) siRNA to p22 phox (siRNA p22 phox ) or control, scrambled siRNA (scramble) as described in . 48h after transfection, total proteins were extracted and subjected to immunoblot analysis of p22 phox . The histograms show the mean +/- SEM values relative to scramble obtained by densitometric analysis of p22 phox bands normalized for α-tubulin of three independent experiments. *p< 0.01 vs scramble. ( C ) 24h after transfection cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min. Total proteins were extracted and subjected to immunoblot analysis of DUOX. The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) obtained by densitometric analysis of DUOX bands normalized for α-tubulin of three independent experiments. * p< 0.01 vs Ctr. ( D, E ) 24h after transfection with a mix of the two p22 phox siRNA, cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min, mRNA was extracted and DUOX1 and DUOX2 mRNA levels were analyzed by RT-PCR as described in . The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) of three independent experiments. * p<0.05 vs Ctr; ** p<0.05 vs PDGF stimulated scramble.
    Figure Legend Snippet: ( A ) Identification of NOX expressed in neuroblastoma (SK-N-BE) and colon carcinoma (Caco-2) cells. Total RNA was extracted with Trizol, reverse transcribed and analyzed by PCR (see ) with specific primers to NOX1, NOX2 and NOX4 (left) or NOX3 and NOX5 (right). The number of cycles was 35. ( B ) Cells were transfected by electroporation with two different (45 and 46) siRNA to p22 phox (siRNA p22 phox ) or control, scrambled siRNA (scramble) as described in . 48h after transfection, total proteins were extracted and subjected to immunoblot analysis of p22 phox . The histograms show the mean +/- SEM values relative to scramble obtained by densitometric analysis of p22 phox bands normalized for α-tubulin of three independent experiments. *p< 0.01 vs scramble. ( C ) 24h after transfection cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min. Total proteins were extracted and subjected to immunoblot analysis of DUOX. The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) obtained by densitometric analysis of DUOX bands normalized for α-tubulin of three independent experiments. * p< 0.01 vs Ctr. ( D, E ) 24h after transfection with a mix of the two p22 phox siRNA, cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min, mRNA was extracted and DUOX1 and DUOX2 mRNA levels were analyzed by RT-PCR as described in . The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) of three independent experiments. * p<0.05 vs Ctr; ** p<0.05 vs PDGF stimulated scramble.

    Techniques Used: Transfection, Electroporation, Western Blot, Incubation, Reverse Transcription Polymerase Chain Reaction

    ( A ) Cells were incubated 18h in medium containing 0.2% FBS, loaded with 10µM DCHF-DA in the presence or absence of the intracellular calcium chelator, BAPTA-AM (10µM), and then stimulated with 15ng/ml of PDGF as described in . ROS levels were measured fluorimetrically at the time intervals indicated. Ca ++ -independent ROS were measured in presence of BAPTA-AM. Ca ++ -dependent ROS levels were derived from the assays performed in the presence or absence of BAPTA-AM. Total levels of ROS induced by PDGF were also measured in the absence (Total) or presence of the NADPH oxidase inhibitor AEBSF. Values are Mean +/- SEM of three independent experiments performed in triplicate. * p< 0.01 and ** p< 0.05 vs not stimulated; § p<0.01 vs the corresponding time point of Total (Ctr) curve. ( B ) Cells were transfected by electroporation with siRNA to p22 phox (siRNA p22 phox ) or control, scrambled siRNA (scramble) as described in . 24h after transfection cells were incubated in medium containing 0.2%FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min. An aliquot of cell medium was collected and analyzed for H 2 O 2 levels as described in . The histograms show the mean +/- SEM values of three independent experiments. * p< 0.01 vs Ctr. ** p< 0.01 vs PDGF stimulated scramble.
    Figure Legend Snippet: ( A ) Cells were incubated 18h in medium containing 0.2% FBS, loaded with 10µM DCHF-DA in the presence or absence of the intracellular calcium chelator, BAPTA-AM (10µM), and then stimulated with 15ng/ml of PDGF as described in . ROS levels were measured fluorimetrically at the time intervals indicated. Ca ++ -independent ROS were measured in presence of BAPTA-AM. Ca ++ -dependent ROS levels were derived from the assays performed in the presence or absence of BAPTA-AM. Total levels of ROS induced by PDGF were also measured in the absence (Total) or presence of the NADPH oxidase inhibitor AEBSF. Values are Mean +/- SEM of three independent experiments performed in triplicate. * p< 0.01 and ** p< 0.05 vs not stimulated; § p<0.01 vs the corresponding time point of Total (Ctr) curve. ( B ) Cells were transfected by electroporation with siRNA to p22 phox (siRNA p22 phox ) or control, scrambled siRNA (scramble) as described in . 24h after transfection cells were incubated in medium containing 0.2%FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min. An aliquot of cell medium was collected and analyzed for H 2 O 2 levels as described in . The histograms show the mean +/- SEM values of three independent experiments. * p< 0.01 vs Ctr. ** p< 0.01 vs PDGF stimulated scramble.

    Techniques Used: Incubation, Derivative Assay, Transfection, Electroporation


    Structured Review

    Santa Cruz Biotechnology human p22 phox
    Time course response of PLCγ 1 induction (A) and astragalin suppression of LPS-induced expression or activation of PLCγ 1 (B), PKCβ 2 (C), NADPH oxidases (D) by astragalin in BEAS-2B cells. Cells were treated with 1–20 μM astragalin and then stimulated with 2 μg/ml LPS for 8 h. Cell lysates were prepared for Western blot analysis with a primary antibody against PLCγ1, phospho-PKCβ2, <t>p22</t> <t>phox</t> , and p47 phox . β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 3-5) represent quantitative results of the upper bands obtained from a densitometer Means not sharing a common superscript refer to significant different at P < 0.05.
    Human P22 Phox, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Astragalin inhibits airway eotaxin-1 induction and epithelial apoptosis through modulating oxidative stress-responsive MAPK signaling"

    Article Title: Astragalin inhibits airway eotaxin-1 induction and epithelial apoptosis through modulating oxidative stress-responsive MAPK signaling

    Journal: BMC Pulmonary Medicine

    doi: 10.1186/1471-2466-14-122

    Time course response of PLCγ 1 induction (A) and astragalin suppression of LPS-induced expression or activation of PLCγ 1 (B), PKCβ 2 (C), NADPH oxidases (D) by astragalin in BEAS-2B cells. Cells were treated with 1–20 μM astragalin and then stimulated with 2 μg/ml LPS for 8 h. Cell lysates were prepared for Western blot analysis with a primary antibody against PLCγ1, phospho-PKCβ2, p22 phox , and p47 phox . β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 3-5) represent quantitative results of the upper bands obtained from a densitometer Means not sharing a common superscript refer to significant different at P < 0.05.
    Figure Legend Snippet: Time course response of PLCγ 1 induction (A) and astragalin suppression of LPS-induced expression or activation of PLCγ 1 (B), PKCβ 2 (C), NADPH oxidases (D) by astragalin in BEAS-2B cells. Cells were treated with 1–20 μM astragalin and then stimulated with 2 μg/ml LPS for 8 h. Cell lysates were prepared for Western blot analysis with a primary antibody against PLCγ1, phospho-PKCβ2, p22 phox , and p47 phox . β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 3-5) represent quantitative results of the upper bands obtained from a densitometer Means not sharing a common superscript refer to significant different at P < 0.05.

    Techniques Used: Expressing, Activation Assay, Western Blot

    Suppression of LPS induction of NADPH oxidases by TLR4 blockade in BEAS-2B cells. Cells were treated with 20 μM astragalin or 20 μg/ml OxPAPC and then stimulated with 2 μg/ml LPS for 8 h. Cell lysates were prepared for Western blot analysis with a primary antibody against phospho-PKCβ2, p22 phox , p47 phox , and eotaxin-1. β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 3-4) represent quantitative results of the upper bands obtained from a densitometer. Means not sharing a common superscript refer to significant different at P < 0.05.
    Figure Legend Snippet: Suppression of LPS induction of NADPH oxidases by TLR4 blockade in BEAS-2B cells. Cells were treated with 20 μM astragalin or 20 μg/ml OxPAPC and then stimulated with 2 μg/ml LPS for 8 h. Cell lysates were prepared for Western blot analysis with a primary antibody against phospho-PKCβ2, p22 phox , p47 phox , and eotaxin-1. β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 3-4) represent quantitative results of the upper bands obtained from a densitometer. Means not sharing a common superscript refer to significant different at P < 0.05.

    Techniques Used: Western Blot

    human p22 phox  (Thermo Fisher)


    Bioz Verified Symbol Thermo Fisher is a verified supplier
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    Structured Review

    Thermo Fisher human p22 phox
    ( A–C ) Mouse constructs encoding EROS and gp91 <t>phox</t> were co-transfected into NIH3T3 ( A ), COS-7 ( B ), and HEK293T ( C ) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. ( D–F ) gp91 phox and <t>p22</t> phox expression in HEK293T cells following transfection with the indicated human constructs. ( G ) Left panel: analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in the presence or absence of EROS and treatment with 10 μg/mL cycloheximide. Right panel: quantitation of the cycloheximide assay (mean of four independent experiments; error bars indicate SD) represented as a fold change of gp91 phox in cells expressing gp91 phox and EROS vectors relative to gp91 phox vector alone at 0 hr and normalised to actin expression. Actin and vinculin were used as loading control. ( H ) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector (MW ≈ 41 kDa) and treated with 10 μg/mL cycloheximide. ( I–J ) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox, or both in differentiated PL985 knockout (KO) for p22 phox ( I ) or EROS ( J ). Data are representative of three independent experiments. See also and . Figure 1—source data 1. Raw unedited blots for . Figure 1—source data 2. Raw unedited blots for . Figure 1—source data 3. Raw unedited blots for . Figure 1—source data 4. Uncropped gels used for .
    Human P22 Phox, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    human p22 phox - by Bioz Stars, 2024-07
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    Images

    1) Product Images from "EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling"

    Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

    Journal: eLife

    doi: 10.7554/eLife.76387

    ( A–C ) Mouse constructs encoding EROS and gp91 phox were co-transfected into NIH3T3 ( A ), COS-7 ( B ), and HEK293T ( C ) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. ( D–F ) gp91 phox and p22 phox expression in HEK293T cells following transfection with the indicated human constructs. ( G ) Left panel: analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in the presence or absence of EROS and treatment with 10 μg/mL cycloheximide. Right panel: quantitation of the cycloheximide assay (mean of four independent experiments; error bars indicate SD) represented as a fold change of gp91 phox in cells expressing gp91 phox and EROS vectors relative to gp91 phox vector alone at 0 hr and normalised to actin expression. Actin and vinculin were used as loading control. ( H ) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector (MW ≈ 41 kDa) and treated with 10 μg/mL cycloheximide. ( I–J ) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox, or both in differentiated PL985 knockout (KO) for p22 phox ( I ) or EROS ( J ). Data are representative of three independent experiments. See also and . Figure 1—source data 1. Raw unedited blots for . Figure 1—source data 2. Raw unedited blots for . Figure 1—source data 3. Raw unedited blots for . Figure 1—source data 4. Uncropped gels used for .
    Figure Legend Snippet: ( A–C ) Mouse constructs encoding EROS and gp91 phox were co-transfected into NIH3T3 ( A ), COS-7 ( B ), and HEK293T ( C ) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. ( D–F ) gp91 phox and p22 phox expression in HEK293T cells following transfection with the indicated human constructs. ( G ) Left panel: analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in the presence or absence of EROS and treatment with 10 μg/mL cycloheximide. Right panel: quantitation of the cycloheximide assay (mean of four independent experiments; error bars indicate SD) represented as a fold change of gp91 phox in cells expressing gp91 phox and EROS vectors relative to gp91 phox vector alone at 0 hr and normalised to actin expression. Actin and vinculin were used as loading control. ( H ) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector (MW ≈ 41 kDa) and treated with 10 μg/mL cycloheximide. ( I–J ) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox, or both in differentiated PL985 knockout (KO) for p22 phox ( I ) or EROS ( J ). Data are representative of three independent experiments. See also and . Figure 1—source data 1. Raw unedited blots for . Figure 1—source data 2. Raw unedited blots for . Figure 1—source data 3. Raw unedited blots for . Figure 1—source data 4. Uncropped gels used for .

    Techniques Used: Construct, Transfection, Expressing, Western Blot, Quantitation Assay, Plasmid Preparation, Transduction, Knock-Out

    ( A–D ) Immunoprecipitation (IP) and size-exclusion chromatography (SEC) analysis of protein complexes associated with EROS. ( A ) IP of EROS in HEK293-F cells expressing StrepII-FLAG-tagged EROS, gp91 phox -GFP, and p22 phox with Western blot for gp91 phox . Lysates treated with peptide N-glycosidase F (PNGaseF) or endoglycosidase H (EndoH) served as reference; FG: fully glycosylated; PG: partially glycosylated; NG: non-glycosylated; Tot: total lysate; RT: run through; Elu: eluate. ( B ) SEC profile of EROS-IP eluate indicating protein (280 nm) and heme (414 nm) content. ( C ) Immunoblot analysis of gp91 phox -GFP, EROS-FLAG, and endogenous p22 phox in SEC fractions 9–14 and 15–18. ( D ) SEC profile of EROS eluate from HEK293-F cells expressing EROS-FLAG, gp91 phox, and p22 phox constructs and treated with heme biosynthesis inhibitor succinyl acetone (10 µg/ml). ( E ) IP of StrepII-FLAG-tagged EROS in HEK293-F treated with succinyl acetone. ( F ) Interaction between gp91 phox and EROS assessed through luminescence production in live HEK293T cells expressing the indicated plasmids fused with the large (LgBIT) or small (SmBIT) fragment of the NanoLuc luciferase (see ‘Methods’). Halo Tag (HT)-SmBIT is the negative control; RLU: relative luminescence unit. ( G ) Yeast growth phenotypes obtained with the specified selective media using gp91 phox bait plasmid and EROS prey plasmid. L: leucine; W: tryptophan; H: histidine; DBD: DNA binding domain of Gal4; AD: activation domain of Gal4 (see ‘Methods’). ( H ) EROS localisation in HEK293 cells transfected with EROS construct (top panel; 3D stack) or EROS and Lap2-GFP constructs (bottom panel; single plane), fixed, permeabilised, and labelled with anti-EROS and anti-calnexin antibodies. Scale bar = 5 μm. Data are representative of at least three independent experiments; error bars indicate SEM of triplicates. See also and . Figure 2—source data 1. Raw unedited blots for . Figure 2—source data 2. Uncropped gels used for .
    Figure Legend Snippet: ( A–D ) Immunoprecipitation (IP) and size-exclusion chromatography (SEC) analysis of protein complexes associated with EROS. ( A ) IP of EROS in HEK293-F cells expressing StrepII-FLAG-tagged EROS, gp91 phox -GFP, and p22 phox with Western blot for gp91 phox . Lysates treated with peptide N-glycosidase F (PNGaseF) or endoglycosidase H (EndoH) served as reference; FG: fully glycosylated; PG: partially glycosylated; NG: non-glycosylated; Tot: total lysate; RT: run through; Elu: eluate. ( B ) SEC profile of EROS-IP eluate indicating protein (280 nm) and heme (414 nm) content. ( C ) Immunoblot analysis of gp91 phox -GFP, EROS-FLAG, and endogenous p22 phox in SEC fractions 9–14 and 15–18. ( D ) SEC profile of EROS eluate from HEK293-F cells expressing EROS-FLAG, gp91 phox, and p22 phox constructs and treated with heme biosynthesis inhibitor succinyl acetone (10 µg/ml). ( E ) IP of StrepII-FLAG-tagged EROS in HEK293-F treated with succinyl acetone. ( F ) Interaction between gp91 phox and EROS assessed through luminescence production in live HEK293T cells expressing the indicated plasmids fused with the large (LgBIT) or small (SmBIT) fragment of the NanoLuc luciferase (see ‘Methods’). Halo Tag (HT)-SmBIT is the negative control; RLU: relative luminescence unit. ( G ) Yeast growth phenotypes obtained with the specified selective media using gp91 phox bait plasmid and EROS prey plasmid. L: leucine; W: tryptophan; H: histidine; DBD: DNA binding domain of Gal4; AD: activation domain of Gal4 (see ‘Methods’). ( H ) EROS localisation in HEK293 cells transfected with EROS construct (top panel; 3D stack) or EROS and Lap2-GFP constructs (bottom panel; single plane), fixed, permeabilised, and labelled with anti-EROS and anti-calnexin antibodies. Scale bar = 5 μm. Data are representative of at least three independent experiments; error bars indicate SEM of triplicates. See also and . Figure 2—source data 1. Raw unedited blots for . Figure 2—source data 2. Uncropped gels used for .

    Techniques Used: Immunoprecipitation, Size-exclusion Chromatography, Expressing, Western Blot, Construct, Luciferase, Negative Control, Plasmid Preparation, Binding Assay, Activation Assay, Transfection

    Diagram depicting the role of EROS in gp91 phox biosynthesis and formation of the heterodimer with p22 phox .
    Figure Legend Snippet: Diagram depicting the role of EROS in gp91 phox biosynthesis and formation of the heterodimer with p22 phox .

    Techniques Used:


    Figure Legend Snippet:

    Techniques Used: Knock-Out, Generated, Over Expression

    human neutrophil cytochrome b light chain p22 phox  (Danaher Inc)


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    Danaher Inc human neutrophil cytochrome b light chain p22 phox
    Human Neutrophil Cytochrome B Light Chain P22 Phox, supplied by Danaher Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Structured Review

    Santa Cruz Biotechnology human cyba sirna
    Effect of <t>CYBA</t> or TRPM4 knockdown on cell proliferation of LS174T and HT29 cells measured by CCK-8 cell assay. (A) LS174T cells transfected with <t>siRNA-CYBA</t> ( A ) or siRNA-TRPM4 ( B ) showed similar proliferation compared to siRNA-control transfected cells. HT29 cells transfected with siRNA-CYBA ( C ) or siRNA-TRPM4 ( D ) showed a significant increase in proliferation compared to siRNA-control transfected cells.
    Human Cyba Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Germline Variants of CYBA and TRPM4 Predispose to Familial Colorectal Cancer"

    Article Title: Germline Variants of CYBA and TRPM4 Predispose to Familial Colorectal Cancer

    Journal: Cancers

    doi: 10.3390/cancers14030670

    Effect of CYBA or TRPM4 knockdown on cell proliferation of LS174T and HT29 cells measured by CCK-8 cell assay. (A) LS174T cells transfected with siRNA-CYBA ( A ) or siRNA-TRPM4 ( B ) showed similar proliferation compared to siRNA-control transfected cells. HT29 cells transfected with siRNA-CYBA ( C ) or siRNA-TRPM4 ( D ) showed a significant increase in proliferation compared to siRNA-control transfected cells.
    Figure Legend Snippet: Effect of CYBA or TRPM4 knockdown on cell proliferation of LS174T and HT29 cells measured by CCK-8 cell assay. (A) LS174T cells transfected with siRNA-CYBA ( A ) or siRNA-TRPM4 ( B ) showed similar proliferation compared to siRNA-control transfected cells. HT29 cells transfected with siRNA-CYBA ( C ) or siRNA-TRPM4 ( D ) showed a significant increase in proliferation compared to siRNA-control transfected cells.

    Techniques Used: CCK-8 Assay, Transfection

    human p22 phox cyba  (OriGene)


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    OriGene human p22 phox cyba
    (A-C) Mouse constructs encoding EROS and gp91 <t>phox</t> were co-transfected into NIH3T3 (A) , COS-7 (B) and HEK293T (C) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. (D-F) gp91 phox and <t>p22</t> phox expression in HEK293T cells following transfection with the indicated human constructs. (G) Analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in presence or absence of EROS and treatment with 10μg/mL cycloheximide. Actin and vinculin were used as loading control. (H) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector and treated with 10μg/mL cycloheximide. (I-J) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox or both in differentiated PL985 knock-out for p22 phox (I) or EROS (J) . n: representative of at least 3 independent experiments. See also Figure S1.
    Human P22 Phox Cyba, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "EROS-mediated control of NOX2 and P2X7 biosynthesis"

    Article Title: EROS-mediated control of NOX2 and P2X7 biosynthesis

    Journal: bioRxiv

    doi: 10.1101/2021.09.14.460103

    (A-C) Mouse constructs encoding EROS and gp91 phox were co-transfected into NIH3T3 (A) , COS-7 (B) and HEK293T (C) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. (D-F) gp91 phox and p22 phox expression in HEK293T cells following transfection with the indicated human constructs. (G) Analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in presence or absence of EROS and treatment with 10μg/mL cycloheximide. Actin and vinculin were used as loading control. (H) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector and treated with 10μg/mL cycloheximide. (I-J) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox or both in differentiated PL985 knock-out for p22 phox (I) or EROS (J) . n: representative of at least 3 independent experiments. See also Figure S1.
    Figure Legend Snippet: (A-C) Mouse constructs encoding EROS and gp91 phox were co-transfected into NIH3T3 (A) , COS-7 (B) and HEK293T (C) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. (D-F) gp91 phox and p22 phox expression in HEK293T cells following transfection with the indicated human constructs. (G) Analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in presence or absence of EROS and treatment with 10μg/mL cycloheximide. Actin and vinculin were used as loading control. (H) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector and treated with 10μg/mL cycloheximide. (I-J) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox or both in differentiated PL985 knock-out for p22 phox (I) or EROS (J) . n: representative of at least 3 independent experiments. See also Figure S1.

    Techniques Used: Construct, Transfection, Expressing, Western Blot, Plasmid Preparation, Transduction, Knock-Out

    (A-D) Immunoprecipitation (IP) and size exclusion chromatography (SEC) analysis of protein complexes associated with EROS. (A) IP of EROS in HEK293-F cells expressing StrepII-FLAG-tagged EROS, gp91 phox -GFP and p22 phox with western blot for gp91 phox . Lysates treated with Peptide N-glycosidase F (PNGaseF) or Endoglycosidase H (EndoH) served as reference; PG: partially glycosylated; NG: non-glycosylated; RT: run through (B) SEC profile of EROS-IP eluate indicating protein (280nm) and heme (414nm) content. (C) Immunoblot analysis of gp91 phox -GFP, EROS-FLAG and endogenous p22 phox in SEC fraction 9-14 and 15-18. (D) SEC profile of EROS eluate from HEK293-F cells expressing EROS-FLAG, gp91 phox and p22 phox constructs and treated with heme biosynthesis inhibitor succinyl acetone (10µg/ml). (E) IP of StrepII-FLAG-tagged EROS in HEK293-F treated with succinyl acetone. (F) Interaction between gp91 phox and EROS assessed through luminescence production in live HEK293T cells expressing the indicated plasmids fused with the large (LgBIT) or small (SmBIT) fragment of the NanoLuc luciferase (see methods). Halo Tag (HT)-SmBIT is the negative control; RLU: Relative Luminescence Unit. ( G) Yeast growth phenotypes obtained with the specified selective media using gp91 phox bait plasmid and EROS prey plasmid. DBD: DNA binding domain of Gal4; AD: Activation domain of Gal4 (see methods). (H) EROS localisation in HEK293 cells transfected with EROS construct (top panel; 3D stack) or EROS and Lap2-GFP constructs (bottom panel; single plane), fixed, permeabilised and labelled with anti-EROS and anti-calnexin antibodies. Scale bar= 5μm. n= representative of at least 3 independent experiments. See also Figure S2.
    Figure Legend Snippet: (A-D) Immunoprecipitation (IP) and size exclusion chromatography (SEC) analysis of protein complexes associated with EROS. (A) IP of EROS in HEK293-F cells expressing StrepII-FLAG-tagged EROS, gp91 phox -GFP and p22 phox with western blot for gp91 phox . Lysates treated with Peptide N-glycosidase F (PNGaseF) or Endoglycosidase H (EndoH) served as reference; PG: partially glycosylated; NG: non-glycosylated; RT: run through (B) SEC profile of EROS-IP eluate indicating protein (280nm) and heme (414nm) content. (C) Immunoblot analysis of gp91 phox -GFP, EROS-FLAG and endogenous p22 phox in SEC fraction 9-14 and 15-18. (D) SEC profile of EROS eluate from HEK293-F cells expressing EROS-FLAG, gp91 phox and p22 phox constructs and treated with heme biosynthesis inhibitor succinyl acetone (10µg/ml). (E) IP of StrepII-FLAG-tagged EROS in HEK293-F treated with succinyl acetone. (F) Interaction between gp91 phox and EROS assessed through luminescence production in live HEK293T cells expressing the indicated plasmids fused with the large (LgBIT) or small (SmBIT) fragment of the NanoLuc luciferase (see methods). Halo Tag (HT)-SmBIT is the negative control; RLU: Relative Luminescence Unit. ( G) Yeast growth phenotypes obtained with the specified selective media using gp91 phox bait plasmid and EROS prey plasmid. DBD: DNA binding domain of Gal4; AD: Activation domain of Gal4 (see methods). (H) EROS localisation in HEK293 cells transfected with EROS construct (top panel; 3D stack) or EROS and Lap2-GFP constructs (bottom panel; single plane), fixed, permeabilised and labelled with anti-EROS and anti-calnexin antibodies. Scale bar= 5μm. n= representative of at least 3 independent experiments. See also Figure S2.

    Techniques Used: Immunoprecipitation, Size-exclusion Chromatography, Expressing, Western Blot, Construct, Luciferase, Negative Control, Plasmid Preparation, Binding Assay, Activation Assay, Transfection


    Structured Review

    Abcam rabbit anti human p22 phox
    NOX1 function is important for cell protrusions and cytoskeletal dynamics at the migrating edge of cells. A, Images showing actin staining (red) and nuclei (blue) indicate filopodial and lamellapodial protrusions (white asterisks) at the wound edge of NOX1wt and NOX1mut COS7 cells in scratch wound healing assays. B, Summary graph showing protrusion density (mean distance between protrusions) and protrusion length, mean ± SEM, n = 4 experiments, 40–50 cells analyzed/experiment. **P < 0.05, **P < 0.01. (C) Images showing NOX1wt and NOX1mut cells at the wound edge stained for phosphorylated p190tyr (white), actin (red), pFAK (green), and composite image of actin and pFAK staining. D, Immunohistochemical images of colon tissue from a control patient without inflammation (NOX1wt uninflamed), a control patient with colitis (NOX1wt colitis), and a patient with an NOX1 stop-gain mutation (NOX1mut colitis), stained for phosphorylated p190tyr (red) and nuclei (DAPI, blue). Images representative of 3 stained sections and 5–8 images/section. E, Top, Images showing cells at the wound edge in NOXwt cells stained for <t>p22-PHOX</t> (green), actin (red), and nuclei (DAPI, blue) on the left and <t>p22-PHOX</t> (green) only on the right. Bottom, Summary graph showing p22-PHOX mean fluorescence (arbitrary units) in migrating leader cells versus follower cells in NOXwt cells, n = 3 experiments, 9 images analyzed/experiment.
    Rabbit Anti Human P22 Phox, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "NOX1 Regulates Collective and Planktonic Cell Migration: Insights From Patients With Pediatric-Onset IBD and NOX1 Deficiency"

    Article Title: NOX1 Regulates Collective and Planktonic Cell Migration: Insights From Patients With Pediatric-Onset IBD and NOX1 Deficiency

    Journal: Inflammatory Bowel Diseases

    doi: 10.1093/ibd/izaa017

    NOX1 function is important for cell protrusions and cytoskeletal dynamics at the migrating edge of cells. A, Images showing actin staining (red) and nuclei (blue) indicate filopodial and lamellapodial protrusions (white asterisks) at the wound edge of NOX1wt and NOX1mut COS7 cells in scratch wound healing assays. B, Summary graph showing protrusion density (mean distance between protrusions) and protrusion length, mean ± SEM, n = 4 experiments, 40–50 cells analyzed/experiment. **P < 0.05, **P < 0.01. (C) Images showing NOX1wt and NOX1mut cells at the wound edge stained for phosphorylated p190tyr (white), actin (red), pFAK (green), and composite image of actin and pFAK staining. D, Immunohistochemical images of colon tissue from a control patient without inflammation (NOX1wt uninflamed), a control patient with colitis (NOX1wt colitis), and a patient with an NOX1 stop-gain mutation (NOX1mut colitis), stained for phosphorylated p190tyr (red) and nuclei (DAPI, blue). Images representative of 3 stained sections and 5–8 images/section. E, Top, Images showing cells at the wound edge in NOXwt cells stained for p22-PHOX (green), actin (red), and nuclei (DAPI, blue) on the left and p22-PHOX (green) only on the right. Bottom, Summary graph showing p22-PHOX mean fluorescence (arbitrary units) in migrating leader cells versus follower cells in NOXwt cells, n = 3 experiments, 9 images analyzed/experiment.
    Figure Legend Snippet: NOX1 function is important for cell protrusions and cytoskeletal dynamics at the migrating edge of cells. A, Images showing actin staining (red) and nuclei (blue) indicate filopodial and lamellapodial protrusions (white asterisks) at the wound edge of NOX1wt and NOX1mut COS7 cells in scratch wound healing assays. B, Summary graph showing protrusion density (mean distance between protrusions) and protrusion length, mean ± SEM, n = 4 experiments, 40–50 cells analyzed/experiment. **P < 0.05, **P < 0.01. (C) Images showing NOX1wt and NOX1mut cells at the wound edge stained for phosphorylated p190tyr (white), actin (red), pFAK (green), and composite image of actin and pFAK staining. D, Immunohistochemical images of colon tissue from a control patient without inflammation (NOX1wt uninflamed), a control patient with colitis (NOX1wt colitis), and a patient with an NOX1 stop-gain mutation (NOX1mut colitis), stained for phosphorylated p190tyr (red) and nuclei (DAPI, blue). Images representative of 3 stained sections and 5–8 images/section. E, Top, Images showing cells at the wound edge in NOXwt cells stained for p22-PHOX (green), actin (red), and nuclei (DAPI, blue) on the left and p22-PHOX (green) only on the right. Bottom, Summary graph showing p22-PHOX mean fluorescence (arbitrary units) in migrating leader cells versus follower cells in NOXwt cells, n = 3 experiments, 9 images analyzed/experiment.

    Techniques Used: Staining, Immunohistochemical staining, Mutagenesis, Fluorescence


    Structured Review

    Abcam rabbit anti human p22 phox
    NOX1 function is important for cell protrusions and cytoskeletal dynamics at the migrating edge of cells. A, Images showing actin staining (red) and nuclei (blue) indicate filopodial and lamellapodial protrusions (white asterisks) at the wound edge of NOX1wt and NOX1mut COS7 cells in scratch wound healing assays. B, Summary graph showing protrusion density (mean distance between protrusions) and protrusion length, mean ± SEM, n = 4 experiments, 40–50 cells analyzed/experiment. **P < 0.05, **P < 0.01. (C) Images showing NOX1wt and NOX1mut cells at the wound edge stained for phosphorylated p190tyr (white), actin (red), pFAK (green), and composite image of actin and pFAK staining. D, Immunohistochemical images of colon tissue from a control patient without inflammation (NOX1wt uninflamed), a control patient with colitis (NOX1wt colitis), and a patient with an NOX1 stop-gain mutation (NOX1mut colitis), stained for phosphorylated p190tyr (red) and nuclei (DAPI, blue). Images representative of 3 stained sections and 5–8 images/section. E, Top, Images showing cells at the wound edge in NOXwt cells stained for <t>p22-PHOX</t> (green), actin (red), and nuclei (DAPI, blue) on the left and <t>p22-PHOX</t> (green) only on the right. Bottom, Summary graph showing p22-PHOX mean fluorescence (arbitrary units) in migrating leader cells versus follower cells in NOXwt cells, n = 3 experiments, 9 images analyzed/experiment.
    Rabbit Anti Human P22 Phox, supplied by Abcam, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "NOX1 Regulates Collective and Planktonic Cell Migration: Insights From Patients With Pediatric-Onset IBD and NOX1 Deficiency"

    Article Title: NOX1 Regulates Collective and Planktonic Cell Migration: Insights From Patients With Pediatric-Onset IBD and NOX1 Deficiency

    Journal: Inflammatory Bowel Diseases

    doi: 10.1093/ibd/izaa017

    NOX1 function is important for cell protrusions and cytoskeletal dynamics at the migrating edge of cells. A, Images showing actin staining (red) and nuclei (blue) indicate filopodial and lamellapodial protrusions (white asterisks) at the wound edge of NOX1wt and NOX1mut COS7 cells in scratch wound healing assays. B, Summary graph showing protrusion density (mean distance between protrusions) and protrusion length, mean ± SEM, n = 4 experiments, 40–50 cells analyzed/experiment. **P < 0.05, **P < 0.01. (C) Images showing NOX1wt and NOX1mut cells at the wound edge stained for phosphorylated p190tyr (white), actin (red), pFAK (green), and composite image of actin and pFAK staining. D, Immunohistochemical images of colon tissue from a control patient without inflammation (NOX1wt uninflamed), a control patient with colitis (NOX1wt colitis), and a patient with an NOX1 stop-gain mutation (NOX1mut colitis), stained for phosphorylated p190tyr (red) and nuclei (DAPI, blue). Images representative of 3 stained sections and 5–8 images/section. E, Top, Images showing cells at the wound edge in NOXwt cells stained for p22-PHOX (green), actin (red), and nuclei (DAPI, blue) on the left and p22-PHOX (green) only on the right. Bottom, Summary graph showing p22-PHOX mean fluorescence (arbitrary units) in migrating leader cells versus follower cells in NOXwt cells, n = 3 experiments, 9 images analyzed/experiment.
    Figure Legend Snippet: NOX1 function is important for cell protrusions and cytoskeletal dynamics at the migrating edge of cells. A, Images showing actin staining (red) and nuclei (blue) indicate filopodial and lamellapodial protrusions (white asterisks) at the wound edge of NOX1wt and NOX1mut COS7 cells in scratch wound healing assays. B, Summary graph showing protrusion density (mean distance between protrusions) and protrusion length, mean ± SEM, n = 4 experiments, 40–50 cells analyzed/experiment. **P < 0.05, **P < 0.01. (C) Images showing NOX1wt and NOX1mut cells at the wound edge stained for phosphorylated p190tyr (white), actin (red), pFAK (green), and composite image of actin and pFAK staining. D, Immunohistochemical images of colon tissue from a control patient without inflammation (NOX1wt uninflamed), a control patient with colitis (NOX1wt colitis), and a patient with an NOX1 stop-gain mutation (NOX1mut colitis), stained for phosphorylated p190tyr (red) and nuclei (DAPI, blue). Images representative of 3 stained sections and 5–8 images/section. E, Top, Images showing cells at the wound edge in NOXwt cells stained for p22-PHOX (green), actin (red), and nuclei (DAPI, blue) on the left and p22-PHOX (green) only on the right. Bottom, Summary graph showing p22-PHOX mean fluorescence (arbitrary units) in migrating leader cells versus follower cells in NOXwt cells, n = 3 experiments, 9 images analyzed/experiment.

    Techniques Used: Staining, Immunohistochemical staining, Mutagenesis, Fluorescence


    Structured Review

    Sanquin monoclonal anti human p22 phox antibody
    Influences of co-treatment with ATRA and ellagic acid or urolithin A on transcription of the O 2 − -generating system-related genes. Total RNAs were extracted from ATRA-treated, ATRA plus ellagic acid-treated (A), and ATRA plus urolithin A-treated (B) U937 cells, and mRNA levels of <t>p22-phox,</t> gp91-phox, p40-phox, p47-phox and p67-phox were determined by semiquantitative RT-PCR as in “Materials and methods.” Data calibrated with the internal controls are indicated as percentages of control values (100%) obtained from ATRA-treated U937 cells and represent the averages of three separate experiments. Error bars indicate standard deviation. **, P < 0.01 compared with the data of ATRA-treated cells.
    Monoclonal Anti Human P22 Phox Antibody, supplied by Sanquin, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Ellagic acid and its fermentative derivative urolithin A show reverse effects on the gp91-phox gene expression, resulting in opposite alterations in all- trans retinoic acid-induced superoxide generating activity of U937 cells"

    Article Title: Ellagic acid and its fermentative derivative urolithin A show reverse effects on the gp91-phox gene expression, resulting in opposite alterations in all- trans retinoic acid-induced superoxide generating activity of U937 cells

    Journal: Biochemistry and Biophysics Reports

    doi: 10.1016/j.bbrep.2020.100891

    Influences of co-treatment with ATRA and ellagic acid or urolithin A on transcription of the O 2 − -generating system-related genes. Total RNAs were extracted from ATRA-treated, ATRA plus ellagic acid-treated (A), and ATRA plus urolithin A-treated (B) U937 cells, and mRNA levels of p22-phox, gp91-phox, p40-phox, p47-phox and p67-phox were determined by semiquantitative RT-PCR as in “Materials and methods.” Data calibrated with the internal controls are indicated as percentages of control values (100%) obtained from ATRA-treated U937 cells and represent the averages of three separate experiments. Error bars indicate standard deviation. **, P < 0.01 compared with the data of ATRA-treated cells.
    Figure Legend Snippet: Influences of co-treatment with ATRA and ellagic acid or urolithin A on transcription of the O 2 − -generating system-related genes. Total RNAs were extracted from ATRA-treated, ATRA plus ellagic acid-treated (A), and ATRA plus urolithin A-treated (B) U937 cells, and mRNA levels of p22-phox, gp91-phox, p40-phox, p47-phox and p67-phox were determined by semiquantitative RT-PCR as in “Materials and methods.” Data calibrated with the internal controls are indicated as percentages of control values (100%) obtained from ATRA-treated U937 cells and represent the averages of three separate experiments. Error bars indicate standard deviation. **, P < 0.01 compared with the data of ATRA-treated cells.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Influences of co-treatment with ATRA and ellagic acid or urolithin A on protein levels of the O 2 − -generating system-related factors. (A) Typical immunoblot profiles. Cytosolic (for p40-phox, p47-phox and p67-phox) and membrane (for p22-phox and gp91-phox) fractions were prepared from ATRA-treated (lane 1), ATRA plus ellagic acid-treated (lane 2) and ATRA plus urolithin A-treated (lane 3) U937 cells, and protein levels were determined by immunoblotting as in “Materials and methods.” Human β-actin (for cytosolic fractions) and Na + /K + -ATPase (for membrane fractions) were used as controls. Quantitative data of ATRA plus ellagic acid-treated (B) and ATRA plus urolithin A-treated (C) U937 cells are indicated as percentages of control values (100%) obtained from ATRA-treated U937 cells, and represent the averages of three separate experiments. Error bars indicate standard deviation. **, P < 0.01 compared with the data of ATRA-treated cells.
    Figure Legend Snippet: Influences of co-treatment with ATRA and ellagic acid or urolithin A on protein levels of the O 2 − -generating system-related factors. (A) Typical immunoblot profiles. Cytosolic (for p40-phox, p47-phox and p67-phox) and membrane (for p22-phox and gp91-phox) fractions were prepared from ATRA-treated (lane 1), ATRA plus ellagic acid-treated (lane 2) and ATRA plus urolithin A-treated (lane 3) U937 cells, and protein levels were determined by immunoblotting as in “Materials and methods.” Human β-actin (for cytosolic fractions) and Na + /K + -ATPase (for membrane fractions) were used as controls. Quantitative data of ATRA plus ellagic acid-treated (B) and ATRA plus urolithin A-treated (C) U937 cells are indicated as percentages of control values (100%) obtained from ATRA-treated U937 cells, and represent the averages of three separate experiments. Error bars indicate standard deviation. **, P < 0.01 compared with the data of ATRA-treated cells.

    Techniques Used: Western Blot, Standard Deviation


    Structured Review

    Santa Cruz Biotechnology antibody against full length human p22 phox
    Characterization of p22phox mutant zebrafish as a CGD model. (A) The p22phox sa11978 allele contains a point mutation that causes a premature stop codon (red circle: premature stop, black circle: normal stop) and putative loss of one of three transmembrane domains (highlighted in blue). (B) Western blot analysis using antibody against human p22phox with lysates from <t>p22+/+</t> and <t>p22−/−</t> larvae at 2 dpf. (C) p22−/− and p22+/+ larvae were infected with A. nidulans, A. fumigatus or mock-infected with PBS. Average spore dose: p22+/+ with A. nidulans=63, p22−/− with A. nidulans=62, p22+/+ with A. fumigatus=59, p22−/− with A. fumigatus=64. (D) Wild-type larvae were infected with A. nidulans or A. fumigatus and treated with 10 µM dexamethasone or 0.1% DMSO vehicle control immediately following infection. Average spore dose: A. nidulans=58, A. fumigatus=75. Survival data (C, D) were pooled from three independent replicates. P values and hazard ratios for survival experiments were calculated by Cox proportional hazard regression analysis.
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    Images

    1) Product Images from "Neutrophil phagocyte oxidase activity controls invasive fungal growth and inflammation in zebrafish"

    Article Title: Neutrophil phagocyte oxidase activity controls invasive fungal growth and inflammation in zebrafish

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.236539

    Characterization of p22phox mutant zebrafish as a CGD model. (A) The p22phox sa11978 allele contains a point mutation that causes a premature stop codon (red circle: premature stop, black circle: normal stop) and putative loss of one of three transmembrane domains (highlighted in blue). (B) Western blot analysis using antibody against human p22phox with lysates from p22+/+ and p22−/− larvae at 2 dpf. (C) p22−/− and p22+/+ larvae were infected with A. nidulans, A. fumigatus or mock-infected with PBS. Average spore dose: p22+/+ with A. nidulans=63, p22−/− with A. nidulans=62, p22+/+ with A. fumigatus=59, p22−/− with A. fumigatus=64. (D) Wild-type larvae were infected with A. nidulans or A. fumigatus and treated with 10 µM dexamethasone or 0.1% DMSO vehicle control immediately following infection. Average spore dose: A. nidulans=58, A. fumigatus=75. Survival data (C, D) were pooled from three independent replicates. P values and hazard ratios for survival experiments were calculated by Cox proportional hazard regression analysis.
    Figure Legend Snippet: Characterization of p22phox mutant zebrafish as a CGD model. (A) The p22phox sa11978 allele contains a point mutation that causes a premature stop codon (red circle: premature stop, black circle: normal stop) and putative loss of one of three transmembrane domains (highlighted in blue). (B) Western blot analysis using antibody against human p22phox with lysates from p22+/+ and p22−/− larvae at 2 dpf. (C) p22−/− and p22+/+ larvae were infected with A. nidulans, A. fumigatus or mock-infected with PBS. Average spore dose: p22+/+ with A. nidulans=63, p22−/− with A. nidulans=62, p22+/+ with A. fumigatus=59, p22−/− with A. fumigatus=64. (D) Wild-type larvae were infected with A. nidulans or A. fumigatus and treated with 10 µM dexamethasone or 0.1% DMSO vehicle control immediately following infection. Average spore dose: A. nidulans=58, A. fumigatus=75. Survival data (C, D) were pooled from three independent replicates. P values and hazard ratios for survival experiments were calculated by Cox proportional hazard regression analysis.

    Techniques Used: Mutagenesis, Western Blot, Infection

    p22−/− larvae infected with A. nidulans have higher fungal burden and increased neutrophil recruitment. Neutrophil-labeled (mpx:gfp) p22+/− and p22−/− larvae were infected with RFP-expressing A. nidulans and imaged by confocal microscopy. (A) Representative images of the same larvae imaged throughout the 96 h experiment. Images represent MIPs of image z-stacks. Scale bar: 20 µm. (B) Heat map representing germination and hyphal growth status in infected larvae. Data are compiled from five independent replicates. Each row represents an individual larva (p22+/− n=43, p22−/− n=34). (C) Mean percentage of larvae containing germinated spores, pooled from four independent replicates. (D) Mean percentage of larvae containing invasive fungal growth as determined by the presence of hyphal branching, pooled from five independent replicates. Data in C and D represent means±s.d. and P values were calculated by t-tests. (E) Bars represent pooled LSmeans±s.e.m. of 2D fungal area from five independent replicates. At 96 hpi, only (13/34) p22−/− larvae remained alive (see B); therefore, data from that time point were excluded from C–E to better represent the group as a whole. (F) Quantification of neutrophil recruitment in the hindbrain of infected larvae. Data represent pooled LSmeans±s.e.m. of neutrophil counts from five independent replicates. P values were calculated by ANOVA with Tukey's multiple comparisons. *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001. n.d., no data.
    Figure Legend Snippet: p22−/− larvae infected with A. nidulans have higher fungal burden and increased neutrophil recruitment. Neutrophil-labeled (mpx:gfp) p22+/− and p22−/− larvae were infected with RFP-expressing A. nidulans and imaged by confocal microscopy. (A) Representative images of the same larvae imaged throughout the 96 h experiment. Images represent MIPs of image z-stacks. Scale bar: 20 µm. (B) Heat map representing germination and hyphal growth status in infected larvae. Data are compiled from five independent replicates. Each row represents an individual larva (p22+/− n=43, p22−/− n=34). (C) Mean percentage of larvae containing germinated spores, pooled from four independent replicates. (D) Mean percentage of larvae containing invasive fungal growth as determined by the presence of hyphal branching, pooled from five independent replicates. Data in C and D represent means±s.d. and P values were calculated by t-tests. (E) Bars represent pooled LSmeans±s.e.m. of 2D fungal area from five independent replicates. At 96 hpi, only (13/34) p22−/− larvae remained alive (see B); therefore, data from that time point were excluded from C–E to better represent the group as a whole. (F) Quantification of neutrophil recruitment in the hindbrain of infected larvae. Data represent pooled LSmeans±s.e.m. of neutrophil counts from five independent replicates. P values were calculated by ANOVA with Tukey's multiple comparisons. *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001. n.d., no data.

    Techniques Used: Infection, Labeling, Expressing, Confocal Microscopy

    A. nidulans induces increased cytokine expression in p22−/− larvae. RT-qPCR to analyze gene expression of pro-inflammatory cytokines (il1b, il6, cxcl8-l1, cxcl8-l2 and tnfa) in p22+/+ and p22−/− larvae. (A) Bars represent the mean fold-change in expression from pooled, uninfected larvae at 3 dpf from three independent replicates as calculated by the ΔΔCq method. (B) Bars represent the mean fold-change in expression pooled from individual larvae infected with A. nidulans at 24 hpi. (C) Heat map representing the relative fold-change in expression in individual larvae (from pooled data represented in B) from three independent replicates as calculated by the ΔΔCq method. Each row represents an individual larva. (p22+/+ n=30, p22−/− n=33). Average spore dose: p22−/−=77, p22+/+=75. P values for each cytokine were calculated by t-tests.
    Figure Legend Snippet: A. nidulans induces increased cytokine expression in p22−/− larvae. RT-qPCR to analyze gene expression of pro-inflammatory cytokines (il1b, il6, cxcl8-l1, cxcl8-l2 and tnfa) in p22+/+ and p22−/− larvae. (A) Bars represent the mean fold-change in expression from pooled, uninfected larvae at 3 dpf from three independent replicates as calculated by the ΔΔCq method. (B) Bars represent the mean fold-change in expression pooled from individual larvae infected with A. nidulans at 24 hpi. (C) Heat map representing the relative fold-change in expression in individual larvae (from pooled data represented in B) from three independent replicates as calculated by the ΔΔCq method. Each row represents an individual larva. (p22+/+ n=30, p22−/− n=33). Average spore dose: p22−/−=77, p22+/+=75. P values for each cytokine were calculated by t-tests.

    Techniques Used: Expressing, Quantitative RT-PCR, Infection

    Neutrophil-specific expression of p22phox rescues invasive fungal growth and excessive inflammation. p22−/− larvae that re-express functional p22phox in neutrophils (p22−/−; mpx:p22:gfp) and neutrophil-labeled p22−/− larvae (p22−/−; mpx:gfp) were infected with RFP-expressing A. nidulans, and imaged by confocal microscopy up to 96 hpi. Note that analysis of p22 neutrophil-specific rescue larvae was performed in the same experiments described in Fig. 4 and that the p22−/− data in Fig. 4 are repeated here for comparison with the neutrophil-specific rescue line. (A) Images represent MIPs of image z-stacks. Scale bar: 20 µm. (B) Heat map representing germination and hyphal growth status in infected larvae from five independent replicates. Each row represents an individual larva (p22−/− neutrophil rescue n=27, p22−/− n=34). (C) Mean percentage of larvae containing germinated spores, pooled from five independent replicates. (D) Mean percentage of larvae containing invasive fungal growth as determined by the presence of hyphal branching, pooled from five independent replicates. Data from C and D represent means±s.d., P values were calculated by t-tests. (E) Bars represent pooled LSmeans±s.e.m. of 2D fungal area from five independent replicates. At 96 hpi, only (13/34) p22−/− larvae remained alive (see B); therefore, data from that time point were excluded from (C–E) to better represent the group as a whole. (F) Quantification of neutrophil recruitment in the hindbrain of infected larvae. Data represent pooled LSmeans±s.e.m. of neutrophil counts from five independent replicates. P values calculated by ANOVA with Tukey's multiple comparisons.*P<0.05, **P<0.01 ****P<0.0001. n.d., no data.
    Figure Legend Snippet: Neutrophil-specific expression of p22phox rescues invasive fungal growth and excessive inflammation. p22−/− larvae that re-express functional p22phox in neutrophils (p22−/−; mpx:p22:gfp) and neutrophil-labeled p22−/− larvae (p22−/−; mpx:gfp) were infected with RFP-expressing A. nidulans, and imaged by confocal microscopy up to 96 hpi. Note that analysis of p22 neutrophil-specific rescue larvae was performed in the same experiments described in Fig. 4 and that the p22−/− data in Fig. 4 are repeated here for comparison with the neutrophil-specific rescue line. (A) Images represent MIPs of image z-stacks. Scale bar: 20 µm. (B) Heat map representing germination and hyphal growth status in infected larvae from five independent replicates. Each row represents an individual larva (p22−/− neutrophil rescue n=27, p22−/− n=34). (C) Mean percentage of larvae containing germinated spores, pooled from five independent replicates. (D) Mean percentage of larvae containing invasive fungal growth as determined by the presence of hyphal branching, pooled from five independent replicates. Data from C and D represent means±s.d., P values were calculated by t-tests. (E) Bars represent pooled LSmeans±s.e.m. of 2D fungal area from five independent replicates. At 96 hpi, only (13/34) p22−/− larvae remained alive (see B); therefore, data from that time point were excluded from (C–E) to better represent the group as a whole. (F) Quantification of neutrophil recruitment in the hindbrain of infected larvae. Data represent pooled LSmeans±s.e.m. of neutrophil counts from five independent replicates. P values calculated by ANOVA with Tukey's multiple comparisons.*P<0.05, **P<0.01 ****P<0.0001. n.d., no data.

    Techniques Used: Expressing, Functional Assay, Labeling, Infection, Confocal Microscopy

    Neutrophil-specific expression of p22phox rescues host survival. p22−/− larvae that re-express functional p22phox in neutrophils (p22−/−; mpx:p22:gfp) and neutrophil-labeled p22−/− larvae (p22−/−; mpx:gfp) were infected with RFP-expressing A. nidulans, and imaged by confocal microscopy up to 96 hpi. (A) Images represent depth-encoded MIPs of image z-stacks at 96 hpi. Color scale represents z-depth and location in the hindbrain in a 140 µm image z-stack. Arrowheads indicate neutrophils occupying the same z-plane as hyphae. (B) Survival analysis of neutrophil-labeled p22−/− and p22−/− neutrophil rescue larvae infected with A. nidulans. Average spore dose: p22−/−=77, p22−/− neutrophil rescue=83. P values and hazard ratios calculated by Cox proportional hazard regression analysis.
    Figure Legend Snippet: Neutrophil-specific expression of p22phox rescues host survival. p22−/− larvae that re-express functional p22phox in neutrophils (p22−/−; mpx:p22:gfp) and neutrophil-labeled p22−/− larvae (p22−/−; mpx:gfp) were infected with RFP-expressing A. nidulans, and imaged by confocal microscopy up to 96 hpi. (A) Images represent depth-encoded MIPs of image z-stacks at 96 hpi. Color scale represents z-depth and location in the hindbrain in a 140 µm image z-stack. Arrowheads indicate neutrophils occupying the same z-plane as hyphae. (B) Survival analysis of neutrophil-labeled p22−/− and p22−/− neutrophil rescue larvae infected with A. nidulans. Average spore dose: p22−/−=77, p22−/− neutrophil rescue=83. P values and hazard ratios calculated by Cox proportional hazard regression analysis.

    Techniques Used: Expressing, Functional Assay, Labeling, Infection, Confocal Microscopy

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  • 91
    OriGene human p22 phox cyba
    (A-C) Mouse constructs encoding EROS and gp91 <t>phox</t> were co-transfected into NIH3T3 (A) , COS-7 (B) and HEK293T (C) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. (D-F) gp91 phox and <t>p22</t> phox expression in HEK293T cells following transfection with the indicated human constructs. (G) Analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in presence or absence of EROS and treatment with 10μg/mL cycloheximide. Actin and vinculin were used as loading control. (H) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector and treated with 10μg/mL cycloheximide. (I-J) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox or both in differentiated PL985 knock-out for p22 phox (I) or EROS (J) . n: representative of at least 3 independent experiments. See also Figure S1.
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    Thermo Fisher human p22 phox sirna
    ( A ) Identification of NOX expressed in neuroblastoma (SK-N-BE) and colon carcinoma (Caco-2) cells. Total RNA was extracted with Trizol, reverse transcribed and analyzed by PCR (see ) with specific primers to NOX1, NOX2 and NOX4 (left) or NOX3 and NOX5 (right). The number of cycles was 35. ( B ) Cells were transfected by electroporation with two different (45 and 46) <t>siRNA</t> to <t>p22</t> <t>phox</t> (siRNA p22 phox ) or control, scrambled siRNA (scramble) as described in . 48h after transfection, total proteins were extracted and subjected to immunoblot analysis of p22 phox . The histograms show the mean +/- SEM values relative to scramble obtained by densitometric analysis of p22 phox bands normalized for α-tubulin of three independent experiments. *p< 0.01 vs scramble. ( C ) 24h after transfection cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min. Total proteins were extracted and subjected to immunoblot analysis of DUOX. The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) obtained by densitometric analysis of DUOX bands normalized for α-tubulin of three independent experiments. * p< 0.01 vs Ctr. ( D, E ) 24h after transfection with a mix of the two p22 phox siRNA, cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min, mRNA was extracted and DUOX1 and DUOX2 mRNA levels were analyzed by RT-PCR as described in . The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) of three independent experiments. * p<0.05 vs Ctr; ** p<0.05 vs PDGF stimulated scramble.
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    Santa Cruz Biotechnology human p22 phox
    Time course response of PLCγ 1 induction (A) and astragalin suppression of LPS-induced expression or activation of PLCγ 1 (B), PKCβ 2 (C), NADPH oxidases (D) by astragalin in BEAS-2B cells. Cells were treated with 1–20 μM astragalin and then stimulated with 2 μg/ml LPS for 8 h. Cell lysates were prepared for Western blot analysis with a primary antibody against PLCγ1, phospho-PKCβ2, <t>p22</t> <t>phox</t> , and p47 phox . β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 3-5) represent quantitative results of the upper bands obtained from a densitometer Means not sharing a common superscript refer to significant different at P < 0.05.
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    Thermo Fisher human p22 phox
    ( A–C ) Mouse constructs encoding EROS and gp91 <t>phox</t> were co-transfected into NIH3T3 ( A ), COS-7 ( B ), and HEK293T ( C ) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. ( D–F ) gp91 phox and <t>p22</t> phox expression in HEK293T cells following transfection with the indicated human constructs. ( G ) Left panel: analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in the presence or absence of EROS and treatment with 10 μg/mL cycloheximide. Right panel: quantitation of the cycloheximide assay (mean of four independent experiments; error bars indicate SD) represented as a fold change of gp91 phox in cells expressing gp91 phox and EROS vectors relative to gp91 phox vector alone at 0 hr and normalised to actin expression. Actin and vinculin were used as loading control. ( H ) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector (MW ≈ 41 kDa) and treated with 10 μg/mL cycloheximide. ( I–J ) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox, or both in differentiated PL985 knockout (KO) for p22 phox ( I ) or EROS ( J ). Data are representative of three independent experiments. See also and . Figure 1—source data 1. Raw unedited blots for . Figure 1—source data 2. Raw unedited blots for . Figure 1—source data 3. Raw unedited blots for . Figure 1—source data 4. Uncropped gels used for .
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    Danaher Inc human neutrophil cytochrome b light chain p22 phox
    ( A–C ) Mouse constructs encoding EROS and gp91 <t>phox</t> were co-transfected into NIH3T3 ( A ), COS-7 ( B ), and HEK293T ( C ) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. ( D–F ) gp91 phox and <t>p22</t> phox expression in HEK293T cells following transfection with the indicated human constructs. ( G ) Left panel: analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in the presence or absence of EROS and treatment with 10 μg/mL cycloheximide. Right panel: quantitation of the cycloheximide assay (mean of four independent experiments; error bars indicate SD) represented as a fold change of gp91 phox in cells expressing gp91 phox and EROS vectors relative to gp91 phox vector alone at 0 hr and normalised to actin expression. Actin and vinculin were used as loading control. ( H ) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector (MW ≈ 41 kDa) and treated with 10 μg/mL cycloheximide. ( I–J ) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox, or both in differentiated PL985 knockout (KO) for p22 phox ( I ) or EROS ( J ). Data are representative of three independent experiments. See also and . Figure 1—source data 1. Raw unedited blots for . Figure 1—source data 2. Raw unedited blots for . Figure 1—source data 3. Raw unedited blots for . Figure 1—source data 4. Uncropped gels used for .
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    Santa Cruz Biotechnology human cyba sirna
    Effect of <t>CYBA</t> or TRPM4 knockdown on cell proliferation of LS174T and HT29 cells measured by CCK-8 cell assay. (A) LS174T cells transfected with <t>siRNA-CYBA</t> ( A ) or siRNA-TRPM4 ( B ) showed similar proliferation compared to siRNA-control transfected cells. HT29 cells transfected with siRNA-CYBA ( C ) or siRNA-TRPM4 ( D ) showed a significant increase in proliferation compared to siRNA-control transfected cells.
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    Abcam rabbit anti human p22 phox
    NOX1 function is important for cell protrusions and cytoskeletal dynamics at the migrating edge of cells. A, Images showing actin staining (red) and nuclei (blue) indicate filopodial and lamellapodial protrusions (white asterisks) at the wound edge of NOX1wt and NOX1mut COS7 cells in scratch wound healing assays. B, Summary graph showing protrusion density (mean distance between protrusions) and protrusion length, mean ± SEM, n = 4 experiments, 40–50 cells analyzed/experiment. **P < 0.05, **P < 0.01. (C) Images showing NOX1wt and NOX1mut cells at the wound edge stained for phosphorylated p190tyr (white), actin (red), pFAK (green), and composite image of actin and pFAK staining. D, Immunohistochemical images of colon tissue from a control patient without inflammation (NOX1wt uninflamed), a control patient with colitis (NOX1wt colitis), and a patient with an NOX1 stop-gain mutation (NOX1mut colitis), stained for phosphorylated p190tyr (red) and nuclei (DAPI, blue). Images representative of 3 stained sections and 5–8 images/section. E, Top, Images showing cells at the wound edge in NOXwt cells stained for <t>p22-PHOX</t> (green), actin (red), and nuclei (DAPI, blue) on the left and <t>p22-PHOX</t> (green) only on the right. Bottom, Summary graph showing p22-PHOX mean fluorescence (arbitrary units) in migrating leader cells versus follower cells in NOXwt cells, n = 3 experiments, 9 images analyzed/experiment.
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    Sanquin monoclonal anti human p22 phox antibody
    Influences of co-treatment with ATRA and ellagic acid or urolithin A on transcription of the O 2 − -generating system-related genes. Total RNAs were extracted from ATRA-treated, ATRA plus ellagic acid-treated (A), and ATRA plus urolithin A-treated (B) U937 cells, and mRNA levels of <t>p22-phox,</t> gp91-phox, p40-phox, p47-phox and p67-phox were determined by semiquantitative RT-PCR as in “Materials and methods.” Data calibrated with the internal controls are indicated as percentages of control values (100%) obtained from ATRA-treated U937 cells and represent the averages of three separate experiments. Error bars indicate standard deviation. **, P < 0.01 compared with the data of ATRA-treated cells.
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    Santa Cruz Biotechnology antibody against full length human p22 phox
    Characterization of p22phox mutant zebrafish as a CGD model. (A) The p22phox sa11978 allele contains a point mutation that causes a premature stop codon (red circle: premature stop, black circle: normal stop) and putative loss of one of three transmembrane domains (highlighted in blue). (B) Western blot analysis using antibody against human p22phox with lysates from <t>p22+/+</t> and <t>p22−/−</t> larvae at 2 dpf. (C) p22−/− and p22+/+ larvae were infected with A. nidulans, A. fumigatus or mock-infected with PBS. Average spore dose: p22+/+ with A. nidulans=63, p22−/− with A. nidulans=62, p22+/+ with A. fumigatus=59, p22−/− with A. fumigatus=64. (D) Wild-type larvae were infected with A. nidulans or A. fumigatus and treated with 10 µM dexamethasone or 0.1% DMSO vehicle control immediately following infection. Average spore dose: A. nidulans=58, A. fumigatus=75. Survival data (C, D) were pooled from three independent replicates. P values and hazard ratios for survival experiments were calculated by Cox proportional hazard regression analysis.
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    (A-C) Mouse constructs encoding EROS and gp91 phox were co-transfected into NIH3T3 (A) , COS-7 (B) and HEK293T (C) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. (D-F) gp91 phox and p22 phox expression in HEK293T cells following transfection with the indicated human constructs. (G) Analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in presence or absence of EROS and treatment with 10μg/mL cycloheximide. Actin and vinculin were used as loading control. (H) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector and treated with 10μg/mL cycloheximide. (I-J) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox or both in differentiated PL985 knock-out for p22 phox (I) or EROS (J) . n: representative of at least 3 independent experiments. See also Figure S1.

    Journal: bioRxiv

    Article Title: EROS-mediated control of NOX2 and P2X7 biosynthesis

    doi: 10.1101/2021.09.14.460103

    Figure Lengend Snippet: (A-C) Mouse constructs encoding EROS and gp91 phox were co-transfected into NIH3T3 (A) , COS-7 (B) and HEK293T (C) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. (D-F) gp91 phox and p22 phox expression in HEK293T cells following transfection with the indicated human constructs. (G) Analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in presence or absence of EROS and treatment with 10μg/mL cycloheximide. Actin and vinculin were used as loading control. (H) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector and treated with 10μg/mL cycloheximide. (I-J) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox or both in differentiated PL985 knock-out for p22 phox (I) or EROS (J) . n: representative of at least 3 independent experiments. See also Figure S1.

    Article Snippet: The following plasmids were obtained from Origene: mouse gp91 phox /nox2/cybb (MC204867), mouse GFP-tagged gp91 phox /nox2/cybb (MG208975), mouse EROS/cybc1 (MC201263), human EROS/CYBC1 (SC324452), human gp91 phox /CYBB (SC122091), human p22 phox /CYBA (SC101113), human NOX4 (SC322623), mouse GFP-tagged P2X7 (MR227216), human P2X1 (SC118594) and human P2X4 (SC122124).

    Techniques: Construct, Transfection, Expressing, Western Blot, Plasmid Preparation, Transduction, Knock-Out

    (A-D) Immunoprecipitation (IP) and size exclusion chromatography (SEC) analysis of protein complexes associated with EROS. (A) IP of EROS in HEK293-F cells expressing StrepII-FLAG-tagged EROS, gp91 phox -GFP and p22 phox with western blot for gp91 phox . Lysates treated with Peptide N-glycosidase F (PNGaseF) or Endoglycosidase H (EndoH) served as reference; PG: partially glycosylated; NG: non-glycosylated; RT: run through (B) SEC profile of EROS-IP eluate indicating protein (280nm) and heme (414nm) content. (C) Immunoblot analysis of gp91 phox -GFP, EROS-FLAG and endogenous p22 phox in SEC fraction 9-14 and 15-18. (D) SEC profile of EROS eluate from HEK293-F cells expressing EROS-FLAG, gp91 phox and p22 phox constructs and treated with heme biosynthesis inhibitor succinyl acetone (10µg/ml). (E) IP of StrepII-FLAG-tagged EROS in HEK293-F treated with succinyl acetone. (F) Interaction between gp91 phox and EROS assessed through luminescence production in live HEK293T cells expressing the indicated plasmids fused with the large (LgBIT) or small (SmBIT) fragment of the NanoLuc luciferase (see methods). Halo Tag (HT)-SmBIT is the negative control; RLU: Relative Luminescence Unit. ( G) Yeast growth phenotypes obtained with the specified selective media using gp91 phox bait plasmid and EROS prey plasmid. DBD: DNA binding domain of Gal4; AD: Activation domain of Gal4 (see methods). (H) EROS localisation in HEK293 cells transfected with EROS construct (top panel; 3D stack) or EROS and Lap2-GFP constructs (bottom panel; single plane), fixed, permeabilised and labelled with anti-EROS and anti-calnexin antibodies. Scale bar= 5μm. n= representative of at least 3 independent experiments. See also Figure S2.

    Journal: bioRxiv

    Article Title: EROS-mediated control of NOX2 and P2X7 biosynthesis

    doi: 10.1101/2021.09.14.460103

    Figure Lengend Snippet: (A-D) Immunoprecipitation (IP) and size exclusion chromatography (SEC) analysis of protein complexes associated with EROS. (A) IP of EROS in HEK293-F cells expressing StrepII-FLAG-tagged EROS, gp91 phox -GFP and p22 phox with western blot for gp91 phox . Lysates treated with Peptide N-glycosidase F (PNGaseF) or Endoglycosidase H (EndoH) served as reference; PG: partially glycosylated; NG: non-glycosylated; RT: run through (B) SEC profile of EROS-IP eluate indicating protein (280nm) and heme (414nm) content. (C) Immunoblot analysis of gp91 phox -GFP, EROS-FLAG and endogenous p22 phox in SEC fraction 9-14 and 15-18. (D) SEC profile of EROS eluate from HEK293-F cells expressing EROS-FLAG, gp91 phox and p22 phox constructs and treated with heme biosynthesis inhibitor succinyl acetone (10µg/ml). (E) IP of StrepII-FLAG-tagged EROS in HEK293-F treated with succinyl acetone. (F) Interaction between gp91 phox and EROS assessed through luminescence production in live HEK293T cells expressing the indicated plasmids fused with the large (LgBIT) or small (SmBIT) fragment of the NanoLuc luciferase (see methods). Halo Tag (HT)-SmBIT is the negative control; RLU: Relative Luminescence Unit. ( G) Yeast growth phenotypes obtained with the specified selective media using gp91 phox bait plasmid and EROS prey plasmid. DBD: DNA binding domain of Gal4; AD: Activation domain of Gal4 (see methods). (H) EROS localisation in HEK293 cells transfected with EROS construct (top panel; 3D stack) or EROS and Lap2-GFP constructs (bottom panel; single plane), fixed, permeabilised and labelled with anti-EROS and anti-calnexin antibodies. Scale bar= 5μm. n= representative of at least 3 independent experiments. See also Figure S2.

    Article Snippet: The following plasmids were obtained from Origene: mouse gp91 phox /nox2/cybb (MC204867), mouse GFP-tagged gp91 phox /nox2/cybb (MG208975), mouse EROS/cybc1 (MC201263), human EROS/CYBC1 (SC324452), human gp91 phox /CYBB (SC122091), human p22 phox /CYBA (SC101113), human NOX4 (SC322623), mouse GFP-tagged P2X7 (MR227216), human P2X1 (SC118594) and human P2X4 (SC122124).

    Techniques: Immunoprecipitation, Size-exclusion Chromatography, Expressing, Western Blot, Construct, Luciferase, Negative Control, Plasmid Preparation, Binding Assay, Activation Assay, Transfection

    ( A ) Identification of NOX expressed in neuroblastoma (SK-N-BE) and colon carcinoma (Caco-2) cells. Total RNA was extracted with Trizol, reverse transcribed and analyzed by PCR (see ) with specific primers to NOX1, NOX2 and NOX4 (left) or NOX3 and NOX5 (right). The number of cycles was 35. ( B ) Cells were transfected by electroporation with two different (45 and 46) siRNA to p22 phox (siRNA p22 phox ) or control, scrambled siRNA (scramble) as described in . 48h after transfection, total proteins were extracted and subjected to immunoblot analysis of p22 phox . The histograms show the mean +/- SEM values relative to scramble obtained by densitometric analysis of p22 phox bands normalized for α-tubulin of three independent experiments. *p< 0.01 vs scramble. ( C ) 24h after transfection cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min. Total proteins were extracted and subjected to immunoblot analysis of DUOX. The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) obtained by densitometric analysis of DUOX bands normalized for α-tubulin of three independent experiments. * p< 0.01 vs Ctr. ( D, E ) 24h after transfection with a mix of the two p22 phox siRNA, cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min, mRNA was extracted and DUOX1 and DUOX2 mRNA levels were analyzed by RT-PCR as described in . The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) of three independent experiments. * p<0.05 vs Ctr; ** p<0.05 vs PDGF stimulated scramble.

    Journal: PLoS ONE

    Article Title: Reactive Oxygen Species Regulate the Levels of Dual Oxidase (Duox1-2) in Human Neuroblastoma Cells

    doi: 10.1371/journal.pone.0034405

    Figure Lengend Snippet: ( A ) Identification of NOX expressed in neuroblastoma (SK-N-BE) and colon carcinoma (Caco-2) cells. Total RNA was extracted with Trizol, reverse transcribed and analyzed by PCR (see ) with specific primers to NOX1, NOX2 and NOX4 (left) or NOX3 and NOX5 (right). The number of cycles was 35. ( B ) Cells were transfected by electroporation with two different (45 and 46) siRNA to p22 phox (siRNA p22 phox ) or control, scrambled siRNA (scramble) as described in . 48h after transfection, total proteins were extracted and subjected to immunoblot analysis of p22 phox . The histograms show the mean +/- SEM values relative to scramble obtained by densitometric analysis of p22 phox bands normalized for α-tubulin of three independent experiments. *p< 0.01 vs scramble. ( C ) 24h after transfection cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min. Total proteins were extracted and subjected to immunoblot analysis of DUOX. The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) obtained by densitometric analysis of DUOX bands normalized for α-tubulin of three independent experiments. * p< 0.01 vs Ctr. ( D, E ) 24h after transfection with a mix of the two p22 phox siRNA, cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min, mRNA was extracted and DUOX1 and DUOX2 mRNA levels were analyzed by RT-PCR as described in . The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) of three independent experiments. * p<0.05 vs Ctr; ** p<0.05 vs PDGF stimulated scramble.

    Article Snippet: Human p22 phox siRNA (Stealth RNAi™) were obtained from Invitrogen Corporation (USA).

    Techniques: Transfection, Electroporation, Western Blot, Incubation, Reverse Transcription Polymerase Chain Reaction

    ( A ) Cells were incubated 18h in medium containing 0.2% FBS, loaded with 10µM DCHF-DA in the presence or absence of the intracellular calcium chelator, BAPTA-AM (10µM), and then stimulated with 15ng/ml of PDGF as described in . ROS levels were measured fluorimetrically at the time intervals indicated. Ca ++ -independent ROS were measured in presence of BAPTA-AM. Ca ++ -dependent ROS levels were derived from the assays performed in the presence or absence of BAPTA-AM. Total levels of ROS induced by PDGF were also measured in the absence (Total) or presence of the NADPH oxidase inhibitor AEBSF. Values are Mean +/- SEM of three independent experiments performed in triplicate. * p< 0.01 and ** p< 0.05 vs not stimulated; § p<0.01 vs the corresponding time point of Total (Ctr) curve. ( B ) Cells were transfected by electroporation with siRNA to p22 phox (siRNA p22 phox ) or control, scrambled siRNA (scramble) as described in . 24h after transfection cells were incubated in medium containing 0.2%FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min. An aliquot of cell medium was collected and analyzed for H 2 O 2 levels as described in . The histograms show the mean +/- SEM values of three independent experiments. * p< 0.01 vs Ctr. ** p< 0.01 vs PDGF stimulated scramble.

    Journal: PLoS ONE

    Article Title: Reactive Oxygen Species Regulate the Levels of Dual Oxidase (Duox1-2) in Human Neuroblastoma Cells

    doi: 10.1371/journal.pone.0034405

    Figure Lengend Snippet: ( A ) Cells were incubated 18h in medium containing 0.2% FBS, loaded with 10µM DCHF-DA in the presence or absence of the intracellular calcium chelator, BAPTA-AM (10µM), and then stimulated with 15ng/ml of PDGF as described in . ROS levels were measured fluorimetrically at the time intervals indicated. Ca ++ -independent ROS were measured in presence of BAPTA-AM. Ca ++ -dependent ROS levels were derived from the assays performed in the presence or absence of BAPTA-AM. Total levels of ROS induced by PDGF were also measured in the absence (Total) or presence of the NADPH oxidase inhibitor AEBSF. Values are Mean +/- SEM of three independent experiments performed in triplicate. * p< 0.01 and ** p< 0.05 vs not stimulated; § p<0.01 vs the corresponding time point of Total (Ctr) curve. ( B ) Cells were transfected by electroporation with siRNA to p22 phox (siRNA p22 phox ) or control, scrambled siRNA (scramble) as described in . 24h after transfection cells were incubated in medium containing 0.2%FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min. An aliquot of cell medium was collected and analyzed for H 2 O 2 levels as described in . The histograms show the mean +/- SEM values of three independent experiments. * p< 0.01 vs Ctr. ** p< 0.01 vs PDGF stimulated scramble.

    Article Snippet: Human p22 phox siRNA (Stealth RNAi™) were obtained from Invitrogen Corporation (USA).

    Techniques: Incubation, Derivative Assay, Transfection, Electroporation

    Time course response of PLCγ 1 induction (A) and astragalin suppression of LPS-induced expression or activation of PLCγ 1 (B), PKCβ 2 (C), NADPH oxidases (D) by astragalin in BEAS-2B cells. Cells were treated with 1–20 μM astragalin and then stimulated with 2 μg/ml LPS for 8 h. Cell lysates were prepared for Western blot analysis with a primary antibody against PLCγ1, phospho-PKCβ2, p22 phox , and p47 phox . β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 3-5) represent quantitative results of the upper bands obtained from a densitometer Means not sharing a common superscript refer to significant different at P < 0.05.

    Journal: BMC Pulmonary Medicine

    Article Title: Astragalin inhibits airway eotaxin-1 induction and epithelial apoptosis through modulating oxidative stress-responsive MAPK signaling

    doi: 10.1186/1471-2466-14-122

    Figure Lengend Snippet: Time course response of PLCγ 1 induction (A) and astragalin suppression of LPS-induced expression or activation of PLCγ 1 (B), PKCβ 2 (C), NADPH oxidases (D) by astragalin in BEAS-2B cells. Cells were treated with 1–20 μM astragalin and then stimulated with 2 μg/ml LPS for 8 h. Cell lysates were prepared for Western blot analysis with a primary antibody against PLCγ1, phospho-PKCβ2, p22 phox , and p47 phox . β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 3-5) represent quantitative results of the upper bands obtained from a densitometer Means not sharing a common superscript refer to significant different at P < 0.05.

    Article Snippet: Antibodies against human TLR4, human p22 phox and human p47 phox were purchased from the Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Expressing, Activation Assay, Western Blot

    Suppression of LPS induction of NADPH oxidases by TLR4 blockade in BEAS-2B cells. Cells were treated with 20 μM astragalin or 20 μg/ml OxPAPC and then stimulated with 2 μg/ml LPS for 8 h. Cell lysates were prepared for Western blot analysis with a primary antibody against phospho-PKCβ2, p22 phox , p47 phox , and eotaxin-1. β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 3-4) represent quantitative results of the upper bands obtained from a densitometer. Means not sharing a common superscript refer to significant different at P < 0.05.

    Journal: BMC Pulmonary Medicine

    Article Title: Astragalin inhibits airway eotaxin-1 induction and epithelial apoptosis through modulating oxidative stress-responsive MAPK signaling

    doi: 10.1186/1471-2466-14-122

    Figure Lengend Snippet: Suppression of LPS induction of NADPH oxidases by TLR4 blockade in BEAS-2B cells. Cells were treated with 20 μM astragalin or 20 μg/ml OxPAPC and then stimulated with 2 μg/ml LPS for 8 h. Cell lysates were prepared for Western blot analysis with a primary antibody against phospho-PKCβ2, p22 phox , p47 phox , and eotaxin-1. β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 3-4) represent quantitative results of the upper bands obtained from a densitometer. Means not sharing a common superscript refer to significant different at P < 0.05.

    Article Snippet: Antibodies against human TLR4, human p22 phox and human p47 phox were purchased from the Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Western Blot

    ( A–C ) Mouse constructs encoding EROS and gp91 phox were co-transfected into NIH3T3 ( A ), COS-7 ( B ), and HEK293T ( C ) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. ( D–F ) gp91 phox and p22 phox expression in HEK293T cells following transfection with the indicated human constructs. ( G ) Left panel: analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in the presence or absence of EROS and treatment with 10 μg/mL cycloheximide. Right panel: quantitation of the cycloheximide assay (mean of four independent experiments; error bars indicate SD) represented as a fold change of gp91 phox in cells expressing gp91 phox and EROS vectors relative to gp91 phox vector alone at 0 hr and normalised to actin expression. Actin and vinculin were used as loading control. ( H ) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector (MW ≈ 41 kDa) and treated with 10 μg/mL cycloheximide. ( I–J ) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox, or both in differentiated PL985 knockout (KO) for p22 phox ( I ) or EROS ( J ). Data are representative of three independent experiments. See also and . Figure 1—source data 1. Raw unedited blots for . Figure 1—source data 2. Raw unedited blots for . Figure 1—source data 3. Raw unedited blots for . Figure 1—source data 4. Uncropped gels used for .

    Journal: eLife

    Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

    doi: 10.7554/eLife.76387

    Figure Lengend Snippet: ( A–C ) Mouse constructs encoding EROS and gp91 phox were co-transfected into NIH3T3 ( A ), COS-7 ( B ), and HEK293T ( C ) cell lines. gp91 phox expression was analysed by immunoblotting; arrow indicates gp91 phox band; ns: non-specific band. ( D–F ) gp91 phox and p22 phox expression in HEK293T cells following transfection with the indicated human constructs. ( G ) Left panel: analysis of the stability of the different forms of gp91 phox (indicated by the arrows) following transfection in HEK293T cells in the presence or absence of EROS and treatment with 10 μg/mL cycloheximide. Right panel: quantitation of the cycloheximide assay (mean of four independent experiments; error bars indicate SD) represented as a fold change of gp91 phox in cells expressing gp91 phox and EROS vectors relative to gp91 phox vector alone at 0 hr and normalised to actin expression. Actin and vinculin were used as loading control. ( H ) Stability of endogenous gp91 phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector (MW ≈ 41 kDa) and treated with 10 μg/mL cycloheximide. ( I–J ) gp91 phox expression following lentiviral transduction of EROS-GFP, gp91 phox, or both in differentiated PL985 knockout (KO) for p22 phox ( I ) or EROS ( J ). Data are representative of three independent experiments. See also and . Figure 1—source data 1. Raw unedited blots for . Figure 1—source data 2. Raw unedited blots for . Figure 1—source data 3. Raw unedited blots for . Figure 1—source data 4. Uncropped gels used for .

    Article Snippet: Cells were transfected at 60–80% confluency with 1.6–2 µg of the following constructs: mouse gp91 phox , mouse GFP-tagged gp91 phox , mouse EROS, human EROS, human gp91 phox , human mRFP-tagged gp91 phox , human p22 phox , human NOX4, mouse GFP-tagged P2X7, human P2X1, and human P2X4; using Lipofectamine RNAiMAX (Thermo Fisher) reagent for HEK293T, HEK293, and COS-7 cells and Lipofectamine 2000 (Thermo Fisher) reagent for NIH3T3 cells, following the manufacturer’s recommendation.

    Techniques: Construct, Transfection, Expressing, Western Blot, Quantitation Assay, Plasmid Preparation, Transduction, Knock-Out

    ( A–D ) Immunoprecipitation (IP) and size-exclusion chromatography (SEC) analysis of protein complexes associated with EROS. ( A ) IP of EROS in HEK293-F cells expressing StrepII-FLAG-tagged EROS, gp91 phox -GFP, and p22 phox with Western blot for gp91 phox . Lysates treated with peptide N-glycosidase F (PNGaseF) or endoglycosidase H (EndoH) served as reference; FG: fully glycosylated; PG: partially glycosylated; NG: non-glycosylated; Tot: total lysate; RT: run through; Elu: eluate. ( B ) SEC profile of EROS-IP eluate indicating protein (280 nm) and heme (414 nm) content. ( C ) Immunoblot analysis of gp91 phox -GFP, EROS-FLAG, and endogenous p22 phox in SEC fractions 9–14 and 15–18. ( D ) SEC profile of EROS eluate from HEK293-F cells expressing EROS-FLAG, gp91 phox, and p22 phox constructs and treated with heme biosynthesis inhibitor succinyl acetone (10 µg/ml). ( E ) IP of StrepII-FLAG-tagged EROS in HEK293-F treated with succinyl acetone. ( F ) Interaction between gp91 phox and EROS assessed through luminescence production in live HEK293T cells expressing the indicated plasmids fused with the large (LgBIT) or small (SmBIT) fragment of the NanoLuc luciferase (see ‘Methods’). Halo Tag (HT)-SmBIT is the negative control; RLU: relative luminescence unit. ( G ) Yeast growth phenotypes obtained with the specified selective media using gp91 phox bait plasmid and EROS prey plasmid. L: leucine; W: tryptophan; H: histidine; DBD: DNA binding domain of Gal4; AD: activation domain of Gal4 (see ‘Methods’). ( H ) EROS localisation in HEK293 cells transfected with EROS construct (top panel; 3D stack) or EROS and Lap2-GFP constructs (bottom panel; single plane), fixed, permeabilised, and labelled with anti-EROS and anti-calnexin antibodies. Scale bar = 5 μm. Data are representative of at least three independent experiments; error bars indicate SEM of triplicates. See also and . Figure 2—source data 1. Raw unedited blots for . Figure 2—source data 2. Uncropped gels used for .

    Journal: eLife

    Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

    doi: 10.7554/eLife.76387

    Figure Lengend Snippet: ( A–D ) Immunoprecipitation (IP) and size-exclusion chromatography (SEC) analysis of protein complexes associated with EROS. ( A ) IP of EROS in HEK293-F cells expressing StrepII-FLAG-tagged EROS, gp91 phox -GFP, and p22 phox with Western blot for gp91 phox . Lysates treated with peptide N-glycosidase F (PNGaseF) or endoglycosidase H (EndoH) served as reference; FG: fully glycosylated; PG: partially glycosylated; NG: non-glycosylated; Tot: total lysate; RT: run through; Elu: eluate. ( B ) SEC profile of EROS-IP eluate indicating protein (280 nm) and heme (414 nm) content. ( C ) Immunoblot analysis of gp91 phox -GFP, EROS-FLAG, and endogenous p22 phox in SEC fractions 9–14 and 15–18. ( D ) SEC profile of EROS eluate from HEK293-F cells expressing EROS-FLAG, gp91 phox, and p22 phox constructs and treated with heme biosynthesis inhibitor succinyl acetone (10 µg/ml). ( E ) IP of StrepII-FLAG-tagged EROS in HEK293-F treated with succinyl acetone. ( F ) Interaction between gp91 phox and EROS assessed through luminescence production in live HEK293T cells expressing the indicated plasmids fused with the large (LgBIT) or small (SmBIT) fragment of the NanoLuc luciferase (see ‘Methods’). Halo Tag (HT)-SmBIT is the negative control; RLU: relative luminescence unit. ( G ) Yeast growth phenotypes obtained with the specified selective media using gp91 phox bait plasmid and EROS prey plasmid. L: leucine; W: tryptophan; H: histidine; DBD: DNA binding domain of Gal4; AD: activation domain of Gal4 (see ‘Methods’). ( H ) EROS localisation in HEK293 cells transfected with EROS construct (top panel; 3D stack) or EROS and Lap2-GFP constructs (bottom panel; single plane), fixed, permeabilised, and labelled with anti-EROS and anti-calnexin antibodies. Scale bar = 5 μm. Data are representative of at least three independent experiments; error bars indicate SEM of triplicates. See also and . Figure 2—source data 1. Raw unedited blots for . Figure 2—source data 2. Uncropped gels used for .

    Article Snippet: Cells were transfected at 60–80% confluency with 1.6–2 µg of the following constructs: mouse gp91 phox , mouse GFP-tagged gp91 phox , mouse EROS, human EROS, human gp91 phox , human mRFP-tagged gp91 phox , human p22 phox , human NOX4, mouse GFP-tagged P2X7, human P2X1, and human P2X4; using Lipofectamine RNAiMAX (Thermo Fisher) reagent for HEK293T, HEK293, and COS-7 cells and Lipofectamine 2000 (Thermo Fisher) reagent for NIH3T3 cells, following the manufacturer’s recommendation.

    Techniques: Immunoprecipitation, Size-exclusion Chromatography, Expressing, Western Blot, Construct, Luciferase, Negative Control, Plasmid Preparation, Binding Assay, Activation Assay, Transfection

    Diagram depicting the role of EROS in gp91 phox biosynthesis and formation of the heterodimer with p22 phox .

    Journal: eLife

    Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

    doi: 10.7554/eLife.76387

    Figure Lengend Snippet: Diagram depicting the role of EROS in gp91 phox biosynthesis and formation of the heterodimer with p22 phox .

    Article Snippet: Cells were transfected at 60–80% confluency with 1.6–2 µg of the following constructs: mouse gp91 phox , mouse GFP-tagged gp91 phox , mouse EROS, human EROS, human gp91 phox , human mRFP-tagged gp91 phox , human p22 phox , human NOX4, mouse GFP-tagged P2X7, human P2X1, and human P2X4; using Lipofectamine RNAiMAX (Thermo Fisher) reagent for HEK293T, HEK293, and COS-7 cells and Lipofectamine 2000 (Thermo Fisher) reagent for NIH3T3 cells, following the manufacturer’s recommendation.

    Techniques:

    Journal: eLife

    Article Title: EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

    doi: 10.7554/eLife.76387

    Figure Lengend Snippet:

    Article Snippet: Cells were transfected at 60–80% confluency with 1.6–2 µg of the following constructs: mouse gp91 phox , mouse GFP-tagged gp91 phox , mouse EROS, human EROS, human gp91 phox , human mRFP-tagged gp91 phox , human p22 phox , human NOX4, mouse GFP-tagged P2X7, human P2X1, and human P2X4; using Lipofectamine RNAiMAX (Thermo Fisher) reagent for HEK293T, HEK293, and COS-7 cells and Lipofectamine 2000 (Thermo Fisher) reagent for NIH3T3 cells, following the manufacturer’s recommendation.

    Techniques: Knock-Out, Generated, Over Expression

    Effect of CYBA or TRPM4 knockdown on cell proliferation of LS174T and HT29 cells measured by CCK-8 cell assay. (A) LS174T cells transfected with siRNA-CYBA ( A ) or siRNA-TRPM4 ( B ) showed similar proliferation compared to siRNA-control transfected cells. HT29 cells transfected with siRNA-CYBA ( C ) or siRNA-TRPM4 ( D ) showed a significant increase in proliferation compared to siRNA-control transfected cells.

    Journal: Cancers

    Article Title: Germline Variants of CYBA and TRPM4 Predispose to Familial Colorectal Cancer

    doi: 10.3390/cancers14030670

    Figure Lengend Snippet: Effect of CYBA or TRPM4 knockdown on cell proliferation of LS174T and HT29 cells measured by CCK-8 cell assay. (A) LS174T cells transfected with siRNA-CYBA ( A ) or siRNA-TRPM4 ( B ) showed similar proliferation compared to siRNA-control transfected cells. HT29 cells transfected with siRNA-CYBA ( C ) or siRNA-TRPM4 ( D ) showed a significant increase in proliferation compared to siRNA-control transfected cells.

    Article Snippet: HT-29 and LS174T cells were transfected with human CYBA siRNA (sc-36149, Santa Cruz Biotechnology, INC) to knockdown CYBA or human TRPM4 siRNA (sc-45439, Santa Cruz Biotechnology, Heidelberg, Germany) to knockdown TRPM4, or scrambled siRNA (sc-37007, Santa Cruz Biotechnology, Heidelberg, Germany) as a control.

    Techniques: CCK-8 Assay, Transfection

    NOX1 function is important for cell protrusions and cytoskeletal dynamics at the migrating edge of cells. A, Images showing actin staining (red) and nuclei (blue) indicate filopodial and lamellapodial protrusions (white asterisks) at the wound edge of NOX1wt and NOX1mut COS7 cells in scratch wound healing assays. B, Summary graph showing protrusion density (mean distance between protrusions) and protrusion length, mean ± SEM, n = 4 experiments, 40–50 cells analyzed/experiment. **P < 0.05, **P < 0.01. (C) Images showing NOX1wt and NOX1mut cells at the wound edge stained for phosphorylated p190tyr (white), actin (red), pFAK (green), and composite image of actin and pFAK staining. D, Immunohistochemical images of colon tissue from a control patient without inflammation (NOX1wt uninflamed), a control patient with colitis (NOX1wt colitis), and a patient with an NOX1 stop-gain mutation (NOX1mut colitis), stained for phosphorylated p190tyr (red) and nuclei (DAPI, blue). Images representative of 3 stained sections and 5–8 images/section. E, Top, Images showing cells at the wound edge in NOXwt cells stained for p22-PHOX (green), actin (red), and nuclei (DAPI, blue) on the left and p22-PHOX (green) only on the right. Bottom, Summary graph showing p22-PHOX mean fluorescence (arbitrary units) in migrating leader cells versus follower cells in NOXwt cells, n = 3 experiments, 9 images analyzed/experiment.

    Journal: Inflammatory Bowel Diseases

    Article Title: NOX1 Regulates Collective and Planktonic Cell Migration: Insights From Patients With Pediatric-Onset IBD and NOX1 Deficiency

    doi: 10.1093/ibd/izaa017

    Figure Lengend Snippet: NOX1 function is important for cell protrusions and cytoskeletal dynamics at the migrating edge of cells. A, Images showing actin staining (red) and nuclei (blue) indicate filopodial and lamellapodial protrusions (white asterisks) at the wound edge of NOX1wt and NOX1mut COS7 cells in scratch wound healing assays. B, Summary graph showing protrusion density (mean distance between protrusions) and protrusion length, mean ± SEM, n = 4 experiments, 40–50 cells analyzed/experiment. **P < 0.05, **P < 0.01. (C) Images showing NOX1wt and NOX1mut cells at the wound edge stained for phosphorylated p190tyr (white), actin (red), pFAK (green), and composite image of actin and pFAK staining. D, Immunohistochemical images of colon tissue from a control patient without inflammation (NOX1wt uninflamed), a control patient with colitis (NOX1wt colitis), and a patient with an NOX1 stop-gain mutation (NOX1mut colitis), stained for phosphorylated p190tyr (red) and nuclei (DAPI, blue). Images representative of 3 stained sections and 5–8 images/section. E, Top, Images showing cells at the wound edge in NOXwt cells stained for p22-PHOX (green), actin (red), and nuclei (DAPI, blue) on the left and p22-PHOX (green) only on the right. Bottom, Summary graph showing p22-PHOX mean fluorescence (arbitrary units) in migrating leader cells versus follower cells in NOXwt cells, n = 3 experiments, 9 images analyzed/experiment.

    Article Snippet: Primary antibodies for rabbit anti-human phosphorylation of focal adhesion kinase (p-FAK), rabbit anti-human p22-PHOX (Abcam), mouse anti-human p-P190 (Santa Cruz Biotechnology), and rhodamine-phallodin (Thermo Fisher Scientific, USA) were added and incubated overnight at 4°C.

    Techniques: Staining, Immunohistochemical staining, Mutagenesis, Fluorescence

    Influences of co-treatment with ATRA and ellagic acid or urolithin A on transcription of the O 2 − -generating system-related genes. Total RNAs were extracted from ATRA-treated, ATRA plus ellagic acid-treated (A), and ATRA plus urolithin A-treated (B) U937 cells, and mRNA levels of p22-phox, gp91-phox, p40-phox, p47-phox and p67-phox were determined by semiquantitative RT-PCR as in “Materials and methods.” Data calibrated with the internal controls are indicated as percentages of control values (100%) obtained from ATRA-treated U937 cells and represent the averages of three separate experiments. Error bars indicate standard deviation. **, P < 0.01 compared with the data of ATRA-treated cells.

    Journal: Biochemistry and Biophysics Reports

    Article Title: Ellagic acid and its fermentative derivative urolithin A show reverse effects on the gp91-phox gene expression, resulting in opposite alterations in all- trans retinoic acid-induced superoxide generating activity of U937 cells

    doi: 10.1016/j.bbrep.2020.100891

    Figure Lengend Snippet: Influences of co-treatment with ATRA and ellagic acid or urolithin A on transcription of the O 2 − -generating system-related genes. Total RNAs were extracted from ATRA-treated, ATRA plus ellagic acid-treated (A), and ATRA plus urolithin A-treated (B) U937 cells, and mRNA levels of p22-phox, gp91-phox, p40-phox, p47-phox and p67-phox were determined by semiquantitative RT-PCR as in “Materials and methods.” Data calibrated with the internal controls are indicated as percentages of control values (100%) obtained from ATRA-treated U937 cells and represent the averages of three separate experiments. Error bars indicate standard deviation. **, P < 0.01 compared with the data of ATRA-treated cells.

    Article Snippet: Monoclonal anti-human p22-phox antibody (449) was kindly provided by Dr. Roos and Dr. Verhoeven (Sanquin Research, and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, The Netherlands).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Influences of co-treatment with ATRA and ellagic acid or urolithin A on protein levels of the O 2 − -generating system-related factors. (A) Typical immunoblot profiles. Cytosolic (for p40-phox, p47-phox and p67-phox) and membrane (for p22-phox and gp91-phox) fractions were prepared from ATRA-treated (lane 1), ATRA plus ellagic acid-treated (lane 2) and ATRA plus urolithin A-treated (lane 3) U937 cells, and protein levels were determined by immunoblotting as in “Materials and methods.” Human β-actin (for cytosolic fractions) and Na + /K + -ATPase (for membrane fractions) were used as controls. Quantitative data of ATRA plus ellagic acid-treated (B) and ATRA plus urolithin A-treated (C) U937 cells are indicated as percentages of control values (100%) obtained from ATRA-treated U937 cells, and represent the averages of three separate experiments. Error bars indicate standard deviation. **, P < 0.01 compared with the data of ATRA-treated cells.

    Journal: Biochemistry and Biophysics Reports

    Article Title: Ellagic acid and its fermentative derivative urolithin A show reverse effects on the gp91-phox gene expression, resulting in opposite alterations in all- trans retinoic acid-induced superoxide generating activity of U937 cells

    doi: 10.1016/j.bbrep.2020.100891

    Figure Lengend Snippet: Influences of co-treatment with ATRA and ellagic acid or urolithin A on protein levels of the O 2 − -generating system-related factors. (A) Typical immunoblot profiles. Cytosolic (for p40-phox, p47-phox and p67-phox) and membrane (for p22-phox and gp91-phox) fractions were prepared from ATRA-treated (lane 1), ATRA plus ellagic acid-treated (lane 2) and ATRA plus urolithin A-treated (lane 3) U937 cells, and protein levels were determined by immunoblotting as in “Materials and methods.” Human β-actin (for cytosolic fractions) and Na + /K + -ATPase (for membrane fractions) were used as controls. Quantitative data of ATRA plus ellagic acid-treated (B) and ATRA plus urolithin A-treated (C) U937 cells are indicated as percentages of control values (100%) obtained from ATRA-treated U937 cells, and represent the averages of three separate experiments. Error bars indicate standard deviation. **, P < 0.01 compared with the data of ATRA-treated cells.

    Article Snippet: Monoclonal anti-human p22-phox antibody (449) was kindly provided by Dr. Roos and Dr. Verhoeven (Sanquin Research, and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, The Netherlands).

    Techniques: Western Blot, Standard Deviation

    Characterization of p22phox mutant zebrafish as a CGD model. (A) The p22phox sa11978 allele contains a point mutation that causes a premature stop codon (red circle: premature stop, black circle: normal stop) and putative loss of one of three transmembrane domains (highlighted in blue). (B) Western blot analysis using antibody against human p22phox with lysates from p22+/+ and p22−/− larvae at 2 dpf. (C) p22−/− and p22+/+ larvae were infected with A. nidulans, A. fumigatus or mock-infected with PBS. Average spore dose: p22+/+ with A. nidulans=63, p22−/− with A. nidulans=62, p22+/+ with A. fumigatus=59, p22−/− with A. fumigatus=64. (D) Wild-type larvae were infected with A. nidulans or A. fumigatus and treated with 10 µM dexamethasone or 0.1% DMSO vehicle control immediately following infection. Average spore dose: A. nidulans=58, A. fumigatus=75. Survival data (C, D) were pooled from three independent replicates. P values and hazard ratios for survival experiments were calculated by Cox proportional hazard regression analysis.

    Journal: Journal of Cell Science

    Article Title: Neutrophil phagocyte oxidase activity controls invasive fungal growth and inflammation in zebrafish

    doi: 10.1242/jcs.236539

    Figure Lengend Snippet: Characterization of p22phox mutant zebrafish as a CGD model. (A) The p22phox sa11978 allele contains a point mutation that causes a premature stop codon (red circle: premature stop, black circle: normal stop) and putative loss of one of three transmembrane domains (highlighted in blue). (B) Western blot analysis using antibody against human p22phox with lysates from p22+/+ and p22−/− larvae at 2 dpf. (C) p22−/− and p22+/+ larvae were infected with A. nidulans, A. fumigatus or mock-infected with PBS. Average spore dose: p22+/+ with A. nidulans=63, p22−/− with A. nidulans=62, p22+/+ with A. fumigatus=59, p22−/− with A. fumigatus=64. (D) Wild-type larvae were infected with A. nidulans or A. fumigatus and treated with 10 µM dexamethasone or 0.1% DMSO vehicle control immediately following infection. Average spore dose: A. nidulans=58, A. fumigatus=75. Survival data (C, D) were pooled from three independent replicates. P values and hazard ratios for survival experiments were calculated by Cox proportional hazard regression analysis.

    Article Snippet: Zebrafish p22 phox was detected using an antibody against full-length human p22 phox [p22-phox (FL-195): sc-20781, Santa Cruz Biotechnology, 1:500 dilution].

    Techniques: Mutagenesis, Western Blot, Infection

    p22−/− larvae infected with A. nidulans have higher fungal burden and increased neutrophil recruitment. Neutrophil-labeled (mpx:gfp) p22+/− and p22−/− larvae were infected with RFP-expressing A. nidulans and imaged by confocal microscopy. (A) Representative images of the same larvae imaged throughout the 96 h experiment. Images represent MIPs of image z-stacks. Scale bar: 20 µm. (B) Heat map representing germination and hyphal growth status in infected larvae. Data are compiled from five independent replicates. Each row represents an individual larva (p22+/− n=43, p22−/− n=34). (C) Mean percentage of larvae containing germinated spores, pooled from four independent replicates. (D) Mean percentage of larvae containing invasive fungal growth as determined by the presence of hyphal branching, pooled from five independent replicates. Data in C and D represent means±s.d. and P values were calculated by t-tests. (E) Bars represent pooled LSmeans±s.e.m. of 2D fungal area from five independent replicates. At 96 hpi, only (13/34) p22−/− larvae remained alive (see B); therefore, data from that time point were excluded from C–E to better represent the group as a whole. (F) Quantification of neutrophil recruitment in the hindbrain of infected larvae. Data represent pooled LSmeans±s.e.m. of neutrophil counts from five independent replicates. P values were calculated by ANOVA with Tukey's multiple comparisons. *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001. n.d., no data.

    Journal: Journal of Cell Science

    Article Title: Neutrophil phagocyte oxidase activity controls invasive fungal growth and inflammation in zebrafish

    doi: 10.1242/jcs.236539

    Figure Lengend Snippet: p22−/− larvae infected with A. nidulans have higher fungal burden and increased neutrophil recruitment. Neutrophil-labeled (mpx:gfp) p22+/− and p22−/− larvae were infected with RFP-expressing A. nidulans and imaged by confocal microscopy. (A) Representative images of the same larvae imaged throughout the 96 h experiment. Images represent MIPs of image z-stacks. Scale bar: 20 µm. (B) Heat map representing germination and hyphal growth status in infected larvae. Data are compiled from five independent replicates. Each row represents an individual larva (p22+/− n=43, p22−/− n=34). (C) Mean percentage of larvae containing germinated spores, pooled from four independent replicates. (D) Mean percentage of larvae containing invasive fungal growth as determined by the presence of hyphal branching, pooled from five independent replicates. Data in C and D represent means±s.d. and P values were calculated by t-tests. (E) Bars represent pooled LSmeans±s.e.m. of 2D fungal area from five independent replicates. At 96 hpi, only (13/34) p22−/− larvae remained alive (see B); therefore, data from that time point were excluded from C–E to better represent the group as a whole. (F) Quantification of neutrophil recruitment in the hindbrain of infected larvae. Data represent pooled LSmeans±s.e.m. of neutrophil counts from five independent replicates. P values were calculated by ANOVA with Tukey's multiple comparisons. *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001. n.d., no data.

    Article Snippet: Zebrafish p22 phox was detected using an antibody against full-length human p22 phox [p22-phox (FL-195): sc-20781, Santa Cruz Biotechnology, 1:500 dilution].

    Techniques: Infection, Labeling, Expressing, Confocal Microscopy

    A. nidulans induces increased cytokine expression in p22−/− larvae. RT-qPCR to analyze gene expression of pro-inflammatory cytokines (il1b, il6, cxcl8-l1, cxcl8-l2 and tnfa) in p22+/+ and p22−/− larvae. (A) Bars represent the mean fold-change in expression from pooled, uninfected larvae at 3 dpf from three independent replicates as calculated by the ΔΔCq method. (B) Bars represent the mean fold-change in expression pooled from individual larvae infected with A. nidulans at 24 hpi. (C) Heat map representing the relative fold-change in expression in individual larvae (from pooled data represented in B) from three independent replicates as calculated by the ΔΔCq method. Each row represents an individual larva. (p22+/+ n=30, p22−/− n=33). Average spore dose: p22−/−=77, p22+/+=75. P values for each cytokine were calculated by t-tests.

    Journal: Journal of Cell Science

    Article Title: Neutrophil phagocyte oxidase activity controls invasive fungal growth and inflammation in zebrafish

    doi: 10.1242/jcs.236539

    Figure Lengend Snippet: A. nidulans induces increased cytokine expression in p22−/− larvae. RT-qPCR to analyze gene expression of pro-inflammatory cytokines (il1b, il6, cxcl8-l1, cxcl8-l2 and tnfa) in p22+/+ and p22−/− larvae. (A) Bars represent the mean fold-change in expression from pooled, uninfected larvae at 3 dpf from three independent replicates as calculated by the ΔΔCq method. (B) Bars represent the mean fold-change in expression pooled from individual larvae infected with A. nidulans at 24 hpi. (C) Heat map representing the relative fold-change in expression in individual larvae (from pooled data represented in B) from three independent replicates as calculated by the ΔΔCq method. Each row represents an individual larva. (p22+/+ n=30, p22−/− n=33). Average spore dose: p22−/−=77, p22+/+=75. P values for each cytokine were calculated by t-tests.

    Article Snippet: Zebrafish p22 phox was detected using an antibody against full-length human p22 phox [p22-phox (FL-195): sc-20781, Santa Cruz Biotechnology, 1:500 dilution].

    Techniques: Expressing, Quantitative RT-PCR, Infection

    Neutrophil-specific expression of p22phox rescues invasive fungal growth and excessive inflammation. p22−/− larvae that re-express functional p22phox in neutrophils (p22−/−; mpx:p22:gfp) and neutrophil-labeled p22−/− larvae (p22−/−; mpx:gfp) were infected with RFP-expressing A. nidulans, and imaged by confocal microscopy up to 96 hpi. Note that analysis of p22 neutrophil-specific rescue larvae was performed in the same experiments described in Fig. 4 and that the p22−/− data in Fig. 4 are repeated here for comparison with the neutrophil-specific rescue line. (A) Images represent MIPs of image z-stacks. Scale bar: 20 µm. (B) Heat map representing germination and hyphal growth status in infected larvae from five independent replicates. Each row represents an individual larva (p22−/− neutrophil rescue n=27, p22−/− n=34). (C) Mean percentage of larvae containing germinated spores, pooled from five independent replicates. (D) Mean percentage of larvae containing invasive fungal growth as determined by the presence of hyphal branching, pooled from five independent replicates. Data from C and D represent means±s.d., P values were calculated by t-tests. (E) Bars represent pooled LSmeans±s.e.m. of 2D fungal area from five independent replicates. At 96 hpi, only (13/34) p22−/− larvae remained alive (see B); therefore, data from that time point were excluded from (C–E) to better represent the group as a whole. (F) Quantification of neutrophil recruitment in the hindbrain of infected larvae. Data represent pooled LSmeans±s.e.m. of neutrophil counts from five independent replicates. P values calculated by ANOVA with Tukey's multiple comparisons.*P<0.05, **P<0.01 ****P<0.0001. n.d., no data.

    Journal: Journal of Cell Science

    Article Title: Neutrophil phagocyte oxidase activity controls invasive fungal growth and inflammation in zebrafish

    doi: 10.1242/jcs.236539

    Figure Lengend Snippet: Neutrophil-specific expression of p22phox rescues invasive fungal growth and excessive inflammation. p22−/− larvae that re-express functional p22phox in neutrophils (p22−/−; mpx:p22:gfp) and neutrophil-labeled p22−/− larvae (p22−/−; mpx:gfp) were infected with RFP-expressing A. nidulans, and imaged by confocal microscopy up to 96 hpi. Note that analysis of p22 neutrophil-specific rescue larvae was performed in the same experiments described in Fig. 4 and that the p22−/− data in Fig. 4 are repeated here for comparison with the neutrophil-specific rescue line. (A) Images represent MIPs of image z-stacks. Scale bar: 20 µm. (B) Heat map representing germination and hyphal growth status in infected larvae from five independent replicates. Each row represents an individual larva (p22−/− neutrophil rescue n=27, p22−/− n=34). (C) Mean percentage of larvae containing germinated spores, pooled from five independent replicates. (D) Mean percentage of larvae containing invasive fungal growth as determined by the presence of hyphal branching, pooled from five independent replicates. Data from C and D represent means±s.d., P values were calculated by t-tests. (E) Bars represent pooled LSmeans±s.e.m. of 2D fungal area from five independent replicates. At 96 hpi, only (13/34) p22−/− larvae remained alive (see B); therefore, data from that time point were excluded from (C–E) to better represent the group as a whole. (F) Quantification of neutrophil recruitment in the hindbrain of infected larvae. Data represent pooled LSmeans±s.e.m. of neutrophil counts from five independent replicates. P values calculated by ANOVA with Tukey's multiple comparisons.*P<0.05, **P<0.01 ****P<0.0001. n.d., no data.

    Article Snippet: Zebrafish p22 phox was detected using an antibody against full-length human p22 phox [p22-phox (FL-195): sc-20781, Santa Cruz Biotechnology, 1:500 dilution].

    Techniques: Expressing, Functional Assay, Labeling, Infection, Confocal Microscopy

    Neutrophil-specific expression of p22phox rescues host survival. p22−/− larvae that re-express functional p22phox in neutrophils (p22−/−; mpx:p22:gfp) and neutrophil-labeled p22−/− larvae (p22−/−; mpx:gfp) were infected with RFP-expressing A. nidulans, and imaged by confocal microscopy up to 96 hpi. (A) Images represent depth-encoded MIPs of image z-stacks at 96 hpi. Color scale represents z-depth and location in the hindbrain in a 140 µm image z-stack. Arrowheads indicate neutrophils occupying the same z-plane as hyphae. (B) Survival analysis of neutrophil-labeled p22−/− and p22−/− neutrophil rescue larvae infected with A. nidulans. Average spore dose: p22−/−=77, p22−/− neutrophil rescue=83. P values and hazard ratios calculated by Cox proportional hazard regression analysis.

    Journal: Journal of Cell Science

    Article Title: Neutrophil phagocyte oxidase activity controls invasive fungal growth and inflammation in zebrafish

    doi: 10.1242/jcs.236539

    Figure Lengend Snippet: Neutrophil-specific expression of p22phox rescues host survival. p22−/− larvae that re-express functional p22phox in neutrophils (p22−/−; mpx:p22:gfp) and neutrophil-labeled p22−/− larvae (p22−/−; mpx:gfp) were infected with RFP-expressing A. nidulans, and imaged by confocal microscopy up to 96 hpi. (A) Images represent depth-encoded MIPs of image z-stacks at 96 hpi. Color scale represents z-depth and location in the hindbrain in a 140 µm image z-stack. Arrowheads indicate neutrophils occupying the same z-plane as hyphae. (B) Survival analysis of neutrophil-labeled p22−/− and p22−/− neutrophil rescue larvae infected with A. nidulans. Average spore dose: p22−/−=77, p22−/− neutrophil rescue=83. P values and hazard ratios calculated by Cox proportional hazard regression analysis.

    Article Snippet: Zebrafish p22 phox was detected using an antibody against full-length human p22 phox [p22-phox (FL-195): sc-20781, Santa Cruz Biotechnology, 1:500 dilution].

    Techniques: Expressing, Functional Assay, Labeling, Infection, Confocal Microscopy