novus biotech  (Novus Biologicals)

Journal: Nature Immunology

Article Title: The astrocyte-produced growth factor HB-EGF limits autoimmune CNS pathology

doi: 10.1038/s41590-024-01756-6

Figure Lengend Snippet: a , Principal Component Analysis (PCA) of the CSF abundance of the measured analytes together with clinical parameters (Supplementary Table ; Age, EDSS, Therapy, number of cerebral lesions, number of spinal lesions, optic neuritis, relapse frequency, number of relapses, disease duration (in weeks), cell count CSF) in controls (n = 20), CIS (n = 21), and RRMS (n = 54) patients. b , CSF levels of GAS6, IL-10, LIF, CCL-2, MIF, NGF-β, TGF-α, VEGF-A, NSE, S100B, GFAP, LAP, YKL-40, SCF, Aβ 1-42 , CD44, TRAIL, and NRGN in (n = 20), CIS (n = 21), and RRMS (n = 54) patients. c , logistic regression of CSF HB-EGF levels in CIS (n = 21) vs. non-CIS (n = 74). d , change in concentration of HB-EGF, VEGF-A, NGF-β, CCL-2, NSE, S100B, GFAP, YKL-40, SCF, Aβ 1-42 , CD44, TRAIL, NRGN from baseline (first timepoint) in a RRMS patient (mean time between timepoints is 85 days). e , correlation between HB-EGF levels in the CSF with age, sex, and disease duration in CIS (left) and RRMS (right) patients. CIS n = 21, RRMS n = 54. f , serum concentration of HB-EGF in controls (n = 43), CIS (n = 21), and RRMS (n = 54) patients. g , CSF concentration of HB-EGF in control (n = 20), CIS (n = 21), SPMS (n = 12), and PPMS patients (n = 15) measured by single-plex ELISA. Patient characteristics are provided in Supplementary Table . Data shown as mean ± SD. Unpaired t-test in ( e ), One-way ANOVA with Dunnett’s multiple comparisons test (tested against controls) in ( b , f , g ).

Article Snippet: For the analysis of sHB-EGF in the serum of EAE mice, a commercial HB-EGF ELISA kit was used (Novus Biotech, no. NBP2-62780) according to the manufacturer’s instructions.

Techniques: Cell Counting, Concentration Assay, Control, Enzyme-linked Immunosorbent Assay

Journal: Nature Immunology

Article Title: The astrocyte-produced growth factor HB-EGF limits autoimmune CNS pathology

doi: 10.1038/s41590-024-01756-6

Figure Lengend Snippet: a , schematic of binding domains, flow cytometric quantification, and representative histograms of membranous HB-EGF (antibody 1) and cytoplasmatic HB-EGF (antibody 2) in astrocytes in response to stimulation with TNF-α and IL-1β over 48 hours. n = 4 per timepoint. b , RT-qPCR analysis of Hbegf expression and quantification of soluble HB-EGF (sHB-EGF) in the supernatant of primary mouse astrocyte in response to stimulation with TNF-α and IL-1β over 48 hours. n = 4 per timepoint. d , schematic of stimulation and supernatant sampling, as well as quantification of sHB-EGF ( e ) in primary mouse astrocytes stimulated with TNF-α and IL-1β for 8 hours, followed by extensive washing before supernatant sampling. n = 4/6 per timepoint. f , RT-qPCR analysis of Hif1a expression by ACSA2 + astrocytes following i.c.v. injection of TNF-α and IL-1β or vehicle. n = 3 per group. g , RT-qPCR analysis of Ldha and Ero1l expression as positive controls for HIF1α related signaling in primary mouse astrocytes stimulated with CoCl 2 over 24 hours. n = 4 per timepoint. h , RT-qPCR analysis of HBEGF expression by human astrocytes under pseudohypoxic conditions (CoCl2). n = 2 per group. i , and Enzyme-linked Immunosorbent Assay (ELISA) of soluble HB-EGF (sHB-EGF) in the supernatant of primary mouse astrocytes under pseudohypoxic conditions (CoCl2). n = 4/16 per group. j , schematic of transcriptional competition between HIF1α and AhR. k , representative scatterplots of HB-EGF expression in primary mouse astrocytes (ACSA2 + GFP+) transduced with a control (Gfap::Scrmbl), AhR (Gfap::Ahr), HIF1α (Gfap::Hif1), or HB-EGF (Gfap::Hbegf) targeting CRISPR/Cas9 vector, quantified by intracellular flow cytometry. n = 3 per group. l , representative histograms depicting HB-EGF staining in astrocytes obtained from Gfap::Scrmbl , Gfap::Ahr and Gfap::Hif1a mice during late-stage EAE. m , RT-qPCR analysis of Hbegf, Ahr, Hif1a, and Ldha in ACSA2+ astrocytes in Gfap::Scrmbl , Gfap::Hif1a , and Gfap::Ahr mice. n = 3 per group. Data shown as mean ± SD. One-way ANOVA with Dunett’s multiple comparisons test (tested against control) in ( a , b , c , e , m ), unpaired t -test in ( f , i ), Two-way ANOVA with Sidak’s multiple comparisons test in ( g ).

Article Snippet: For the analysis of sHB-EGF in the serum of EAE mice, a commercial HB-EGF ELISA kit was used (Novus Biotech, no. NBP2-62780) according to the manufacturer’s instructions.

Techniques: Binding Assay, Quantitative RT-PCR, Expressing, Sampling, Injection, Enzyme-linked Immunosorbent Assay, Transduction, Control, CRISPR, Plasmid Preparation, Flow Cytometry, Staining

Journal: Nature Immunology

Article Title: The astrocyte-produced growth factor HB-EGF limits autoimmune CNS pathology

doi: 10.1038/s41590-024-01756-6

Figure Lengend Snippet: a , Principal Component Analysis (PCA) of the CSF abundance of the measured analytes together with clinical parameters (Supplementary Table ; Age, EDSS, Therapy, number of cerebral lesions, number of spinal lesions, optic neuritis, relapse frequency, number of relapses, disease duration (in weeks), cell count CSF) in controls (n = 20), CIS (n = 21), and RRMS (n = 54) patients. b , CSF levels of GAS6, IL-10, LIF, CCL-2, MIF, NGF-β, TGF-α, VEGF-A, NSE, S100B, GFAP, LAP, YKL-40, SCF, Aβ 1-42 , CD44, TRAIL, and NRGN in (n = 20), CIS (n = 21), and RRMS (n = 54) patients. c , logistic regression of CSF HB-EGF levels in CIS (n = 21) vs. non-CIS (n = 74). d , change in concentration of HB-EGF, VEGF-A, NGF-β, CCL-2, NSE, S100B, GFAP, YKL-40, SCF, Aβ 1-42 , CD44, TRAIL, NRGN from baseline (first timepoint) in a RRMS patient (mean time between timepoints is 85 days). e , correlation between HB-EGF levels in the CSF with age, sex, and disease duration in CIS (left) and RRMS (right) patients. CIS n = 21, RRMS n = 54. f , serum concentration of HB-EGF in controls (n = 43), CIS (n = 21), and RRMS (n = 54) patients. g , CSF concentration of HB-EGF in control (n = 20), CIS (n = 21), SPMS (n = 12), and PPMS patients (n = 15) measured by single-plex ELISA. Patient characteristics are provided in Supplementary Table . Data shown as mean ± SD. Unpaired t-test in ( e ), One-way ANOVA with Dunnett’s multiple comparisons test (tested against controls) in ( b , f , g ).

Article Snippet: For the analysis of sHB-EGF in the serum of patients with MS and controls, a commercial HB-EGF ELISA kit was used (Thermo Fisher Scientific, no. EHHBEGFX5) according to the manufacturer’s instructions.

Techniques: Cell Counting, Concentration Assay, Control, Enzyme-linked Immunosorbent Assay

Journal: Nature Immunology

Article Title: The astrocyte-produced growth factor HB-EGF limits autoimmune CNS pathology

doi: 10.1038/s41590-024-01756-6

Figure Lengend Snippet: a , schematic of binding domains, flow cytometric quantification, and representative histograms of membranous HB-EGF (antibody 1) and cytoplasmatic HB-EGF (antibody 2) in astrocytes in response to stimulation with TNF-α and IL-1β over 48 hours. n = 4 per timepoint. b , RT-qPCR analysis of Hbegf expression and quantification of soluble HB-EGF (sHB-EGF) in the supernatant of primary mouse astrocyte in response to stimulation with TNF-α and IL-1β over 48 hours. n = 4 per timepoint. d , schematic of stimulation and supernatant sampling, as well as quantification of sHB-EGF ( e ) in primary mouse astrocytes stimulated with TNF-α and IL-1β for 8 hours, followed by extensive washing before supernatant sampling. n = 4/6 per timepoint. f , RT-qPCR analysis of Hif1a expression by ACSA2 + astrocytes following i.c.v. injection of TNF-α and IL-1β or vehicle. n = 3 per group. g , RT-qPCR analysis of Ldha and Ero1l expression as positive controls for HIF1α related signaling in primary mouse astrocytes stimulated with CoCl 2 over 24 hours. n = 4 per timepoint. h , RT-qPCR analysis of HBEGF expression by human astrocytes under pseudohypoxic conditions (CoCl2). n = 2 per group. i , and Enzyme-linked Immunosorbent Assay (ELISA) of soluble HB-EGF (sHB-EGF) in the supernatant of primary mouse astrocytes under pseudohypoxic conditions (CoCl2). n = 4/16 per group. j , schematic of transcriptional competition between HIF1α and AhR. k , representative scatterplots of HB-EGF expression in primary mouse astrocytes (ACSA2 + GFP+) transduced with a control (Gfap::Scrmbl), AhR (Gfap::Ahr), HIF1α (Gfap::Hif1), or HB-EGF (Gfap::Hbegf) targeting CRISPR/Cas9 vector, quantified by intracellular flow cytometry. n = 3 per group. l , representative histograms depicting HB-EGF staining in astrocytes obtained from Gfap::Scrmbl , Gfap::Ahr and Gfap::Hif1a mice during late-stage EAE. m , RT-qPCR analysis of Hbegf, Ahr, Hif1a, and Ldha in ACSA2+ astrocytes in Gfap::Scrmbl , Gfap::Hif1a , and Gfap::Ahr mice. n = 3 per group. Data shown as mean ± SD. One-way ANOVA with Dunett’s multiple comparisons test (tested against control) in ( a , b , c , e , m ), unpaired t -test in ( f , i ), Two-way ANOVA with Sidak’s multiple comparisons test in ( g ).

Article Snippet: For the analysis of sHB-EGF in the serum of patients with MS and controls, a commercial HB-EGF ELISA kit was used (Thermo Fisher Scientific, no. EHHBEGFX5) according to the manufacturer’s instructions.

Techniques: Binding Assay, Quantitative RT-PCR, Expressing, Sampling, Injection, Enzyme-linked Immunosorbent Assay, Transduction, Control, CRISPR, Plasmid Preparation, Flow Cytometry, Staining