Mouse IFITM3/Fragilis Antibody

Detection of Mouse IFITM3/Fragilis by Western Blot. Western blot shows lysates of NIH-3T3 mouse embryonic fibroblast cell line, D3 mouse embryonic stem cell line, and NTera-2 human testicular embryonic carcinoma cell line. PVDF membrane was probed with 1 µg/mL of Goat Anti-Mouse IFITM3/Fragilis Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3377) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF019). A specific band was detected for IFITM3/Fragilis at approximately 17 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 8.

IFITM3/Fragilis in Embryonic Mouse Ovary. IFITM3/Fragilis was detected in immersion fixed frozen sections of embryonic mouse ovary (E13.5) using 10 µg/mL Goat Anti-Mouse IFITM3/Fragilis Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3377) overnight at 4 °C. Tissue was stained with the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL001) and counterstained with DAPI (blue). View our protocol for Fluorescent IHC Staining of Frozen Tissue Sections.

Detection of Mouse IFITM3/Fragilis by Western Blot Viral replication is required for influenza virus to activate mTORC1 independently of NS1.(A, B) Vero cells were infected with (A) WSN or (B) PR8 wild-type or mutant viruses lacking NS1 at MOI of 2 PFU/cell for 6 h. (C,D) HCT116 Akt1/2+/+ and Akt1/2-/- were infected with WSN delta NS1 or WSN at MOI of 5 PFU/cell for 10h. (E) A549 cells were infected with WSN or UV-inactivated WSN at MOI of 2 PFU/cell for 6h. (F) Mavs+/+ and Mavs-/- or (G) Ifitm3+/+ and Ifitm3-/- MEFs were infected with WSN at an MOI of 2 PFU/cell for 6 h. Immunoblot analyses were performed for detection of viral proteins (NS1, HA, NP and M1) and host proteins ( beta -actin, MAVS, IFITM3 as well as total and phosphorylated S6K, AKT and 4E-BP1). beta -actin and total S6K serve as loading controls. The upper band in the S6K/p-S6K blots is p85 S6K whereas the lower band is p70 S6K. Data are representative of three independent experiments. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.ppat.1006635), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse IFITM3/Fragilis by Western Blot Both interferons and gp130-mediated cytokines induce Ifitm3 expression.Ifitm3 protein expression can also be induced by gp130-family cytokines in cells expressing appropriate receptors. The Ifitm3 promoter alone among murine Ifitm promoters is interferon responsive. (A) Expression of Ifitm3 was determined by western blot in NIH 3T3 and RAW 264.7 cells treated for 2 days with the indicated cytokines. IFN lambda 2 failed to induce Ifitm3 expression at similar concentrations, likely due to lack of the type III IFN receptor. The gp130-family cytokine oncostatin M (OSM) was a potent inducer of Ifitm3 expression in 3T3 cells whereas RAW264.7 cells were responsive to IL-6, but not OSM. (B) Surface expression of the OSM receptor, IL-6 receptor, and gp130 by NIH 3T3 and RAW264.7 cells was determined by flow cytometry. Solid blue lines correspond to the fluorescence signal of the indicated receptor-binding antibodies and red lines show fluorescence of the isotype control. The responsiveness of 3T3 and RAW cells to OSM and IL-6 respectively (as shown in panel A) correlates with their expression of their corresponding receptors. (C) Activities of the six murine Ifitm promoters were assayed in mock-treated, IFN alpha 2-treated, and IFN gamma -treated NIH 3T3 cells transfected with plasmids encoding luciferase under the control of the indicated promoters. Ifitm1, 2, and 6 promoters appear constitutively active, and in contrast to human orthologs, insensitive to interferon stimulation. The Ifitm5 promoter shows almost no activity under any condition, consistent with its tissue-restricted expression in vivo[15]. The Ifitm3 promoter alone is interferon responsive (4.4-fold induction with IFN alpha 2; 2.2-fold with IFN gamma ). Bars show mean of triplicates ± SEM. Asterisk indicates p<0.0001 (two-factor ANOVA Bonferroni post test). (D) Cytokine regulation of Ifitm1, 2, and 3 was confirmed at the mRNA level by qRT-PCR in both 3T3 and RAW cells. Bars depict fold upregulation of the indicated transcripts following a 24-hour incubation with the indicated cytokines relative to transcript levels in untreated cells. Ifitm3 transcript levels were significantly upregulated by IFN alpha 2 (500 ng/ml) and IFN gamma (100 ng/ml) in both cells lines. As in panel (A), OSM (250 ng/ml) upregulated Ifitm3 in 3T3 cells and IL-6 (250 ng/ml) upregulated Ifitm3 in RAW cells. Ifitm1 and Ifitm2 transcript levels were not increased by interferons. Mean Ifitm1 expression increased in response to OSM treatment but the difference was not significant due to the high variance of these samples. Error bars show mean ± range of triplicates. Asterisk indicates p<0.05 and double asterisk indicates p<0.01 (1-tail t-test assuming heteroscedastic samples). Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.ppat.1002909), licensed under a CC-BY license. Not internally tested by R&D Systems.