human p22 phox  (Thermo Fisher)

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

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

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