ifit 3  (Proteintech)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Monosodium Urate Activates Src/Pyk2/PI3 Kinase and Cathepsin Dependent Unconventional Protein Secretion From Human Primary Macrophages *

doi: 10.1074/mcp.M112.024661

Figure Lengend Snippet: The secretion of danger signal proteins, chemokines and interferon-induced proteins is induced on MSU stimulation from LPS-primed macrophages. A, Human macrophages were left unstimulated or primed with LPS for 21 h. After this macrophages were left untreated or activated with MSU for 3 h and the cell culture supernatants were collected. Exosomal fractions were enriched as depicted in Materials and Methods, and Western blotting analysis of danger signal proteins Annexin-1, Galectin-3 (Gal-3), and HSP90 was performed. B, Human macrophages were stimulated as in (A), and the secretion of chemokines CCL2, CCL3, CCL4, CCL5, and CXCL10 was quantified by Luminex assay. Representative data from one experiment is shown; n = 2 independent experiments. C, Macrophages were left untreated or primed with LPS after which they were stimulated with MSU for 3 and 6 h. Total cellular RNA was isolated, and expression of chemokine genes was analyzed with quantitative RT-PCR as depicted in Materials and Methods. The data are reported as fold induction compared with controls. Mean with S.D. of two independent experiments is shown. D, Western blot analysis of IFIT-3 and MxA expression from total cellular lysates and cell culture supernatants of untreated and LPS-primed macrophages with and without MSU stimulation.

Article Snippet: Antibodies against HSP90 (Cell Signaling Technology, Danvers, MA), IFIT-3 (also known as RIG-G) (BD Biosciences), Annexin-1, Cathepsin B (Calbiochem), Cathepsin D, Galectin-3, LAMP-1 (all from Santa Cruz Biotechnology, Santa Cruz, CA), and ASC (Millipore) were purchased.

Techniques: Cell Culture, Western Blot, Luminex, Isolation, Expressing, Quantitative RT-PCR

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Monosodium Urate Activates Src/Pyk2/PI3 Kinase and Cathepsin Dependent Unconventional Protein Secretion From Human Primary Macrophages *

doi: 10.1074/mcp.M112.024661

Figure Lengend Snippet: Cathepsin activity is required for unconventional protein secretion on MSU stimulation. A, Macrophages were left untreated or LPS-primed after which they were activated with MSU for 3 h. Cathepsin B and D expression in total cellular lysates and secretion to supernatants was analyzed with Western blotting. B, Untreated and LPS-primed macrophages were activated with MSU for 3 h in the presence and absence of CA-074 Me. After this IL-1β secretion was measured from supernatants with ELISA. Representative data from one experiment is shown; n = 4 independent experiments. C, Untreated and LPS-primed macrophages were activated with MSU for 3 h in the presence and absence of CA-074 Me. Secreted proteins were visualized by silver staining. D, Secretion of IL-1β, IL-18, ASC, IFIT-3, Galectin-3 (Gal-3), and LAMP-1 was analyzed by Western blotting. E, The secretion of CCL2, CCL3 and CCL4 was quantified with Luminex assay. Representative data from one experiment is shown; n = 4 independent experiments.

Article Snippet: Antibodies against HSP90 (Cell Signaling Technology, Danvers, MA), IFIT-3 (also known as RIG-G) (BD Biosciences), Annexin-1, Cathepsin B (Calbiochem), Cathepsin D, Galectin-3, LAMP-1 (all from Santa Cruz Biotechnology, Santa Cruz, CA), and ASC (Millipore) were purchased.

Techniques: Activity Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Silver Staining, Luminex