goat anti flotillin 1  (Novus Biologicals)

Journal: Cell reports

Article Title: Extracellular vesicles carrying HIV-1 Nef induce long-term hyperreactivity of myeloid cells

doi: 10.1016/j.celrep.2022.111674

Figure Lengend Snippet: (A) Diagram of in vitro experiments in this study. Monocytes treated with exNef or exCont in the presence or absence of methyl-β-cyclodextrin (MβCD), fluvastatin (Flu), or picropodophyllin (PPP) were analyzed by western blotting (WB) for flotillin 1 (Flot1) or by flow cytometry for lipid rafts (staining with CTB) and IGF1R. After differentiation in the presence of M-CSF, MDMs were characterized by t-SNE for activation markers or by ddPCR for gene expression. Following LPS stimulation, gene expression was characterized by RNA-seq and cytokine production measured by ELISA. See also and . (B) Analysis of TNF-α and IL-6 production by LPS-treated MDMs. The graph shows fold increase of cytokine production by exNef-treated over exCont-treated cells from 8 donors (8 biological replicates, 3 technical replicates for each donor), analyzed by Friedman multiple comparison test with Dunn’s correction. Adjusted p values are shown above bars. (C) Dose response analysis. The graph shows mean ± SEM (3 technical replicates of cells from a single donor) fold increase of TNF-α produced by MDMs exposed to exNef with indicated concentrations of Nef versus MDM exposed to exCont at the same concentration of EVs. (D) Gene set enrichment analysis (GSEA) showing normalized enrichment scores for genes differentially expressed between exNef and exCont and participating in the inflammatory response (left panel) and cytokine pathway (right panel); the gene sets were obtained from BioCarta. (E) Leading edge analysis shows that a number of cytokine genes drive the changes of pathways dysregulated by exNef. See also - .

Article Snippet: goat polyclonal anti-human Flotillin 1 , Bio-Techne , cat#NB100-1043.

Techniques: In Vitro, Western Blot, Flow Cytometry, Staining, Activation Assay, Expressing, RNA Sequencing Assay, Enzyme-linked Immunosorbent Assay, Comparison, Produced, Concentration Assay

Journal: Cell reports

Article Title: Extracellular vesicles carrying HIV-1 Nef induce long-term hyperreactivity of myeloid cells

doi: 10.1016/j.celrep.2022.111674

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: goat polyclonal anti-human Flotillin 1 , Bio-Techne , cat#NB100-1043.

Techniques: Virus, Plasmid Preparation, Recombinant, Modification, Staining, RNA HS Assay, SYBR Green Assay, Membrane, Isolation, Cell Fractionation, Lysis, Blocking Assay, Enzyme-linked Immunosorbent Assay, Software, Western Blot

Journal: Cell reports

Article Title: Extracellular vesicles carrying HIV-1 Nef induce long-term hyperreactivity of myeloid cells

doi: 10.1016/j.celrep.2022.111674

Figure Lengend Snippet: (A) Diagram of in vitro experiments in this study. Monocytes treated with exNef or exCont in the presence or absence of methyl-β-cyclodextrin (MβCD), fluvastatin (Flu), or picropodophyllin (PPP) were analyzed by western blotting (WB) for flotillin 1 (Flot1) or by flow cytometry for lipid rafts (staining with CTB) and IGF1R. After differentiation in the presence of M-CSF, MDMs were characterized by t-SNE for activation markers or by ddPCR for gene expression. Following LPS stimulation, gene expression was characterized by RNA-seq and cytokine production measured by ELISA. See also and . (B) Analysis of TNF-α and IL-6 production by LPS-treated MDMs. The graph shows fold increase of cytokine production by exNef-treated over exCont-treated cells from 8 donors (8 biological replicates, 3 technical replicates for each donor), analyzed by Friedman multiple comparison test with Dunn’s correction. Adjusted p values are shown above bars. (C) Dose response analysis. The graph shows mean ± SEM (3 technical replicates of cells from a single donor) fold increase of TNF-α produced by MDMs exposed to exNef with indicated concentrations of Nef versus MDM exposed to exCont at the same concentration of EVs. (D) Gene set enrichment analysis (GSEA) showing normalized enrichment scores for genes differentially expressed between exNef and exCont and participating in the inflammatory response (left panel) and cytokine pathway (right panel); the gene sets were obtained from BioCarta. (E) Leading edge analysis shows that a number of cytokine genes drive the changes of pathways dysregulated by exNef. See also - .

Article Snippet: Capillaries with immobilized proteins were blocked with KPL Detection Block (5X) (SeraCare Life Sciences, Gaithersburg, MD) for 60 min, and then incubated with primary anti-human Flotillin 1 goat polyclonal antibody (Bio-Techne) or anti-hβ-actin mouse monoclonal antibody (R&D) for 60 min. After a wash step, HRP-conjugated anti-goat or near infrared (NIR) fluorescent dye-conjugated anti-mouse secondary antibody from Bio-Techne was added for 30 min to capillaries.

Techniques: In Vitro, Western Blot, Flow Cytometry, Staining, Activation Assay, Expressing, RNA Sequencing Assay, Enzyme-linked Immunosorbent Assay, Comparison, Produced, Concentration Assay

Journal: Cell reports

Article Title: Extracellular vesicles carrying HIV-1 Nef induce long-term hyperreactivity of myeloid cells

doi: 10.1016/j.celrep.2022.111674

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Capillaries with immobilized proteins were blocked with KPL Detection Block (5X) (SeraCare Life Sciences, Gaithersburg, MD) for 60 min, and then incubated with primary anti-human Flotillin 1 goat polyclonal antibody (Bio-Techne) or anti-hβ-actin mouse monoclonal antibody (R&D) for 60 min. After a wash step, HRP-conjugated anti-goat or near infrared (NIR) fluorescent dye-conjugated anti-mouse secondary antibody from Bio-Techne was added for 30 min to capillaries.

Techniques: Virus, Plasmid Preparation, Recombinant, Modification, Staining, RNA HS Assay, SYBR Green Assay, Membrane, Isolation, Cell Fractionation, Lysis, Blocking Assay, Enzyme-linked Immunosorbent Assay, Software, Western Blot