mouse monoclonal anti ifitm2  (Proteintech)

Journal: Frontiers in Microbiology

Article Title: Inhibition of Arenavirus Entry and Replication by the Cell-Intrinsic Restriction Factor ZMPSTE24 Is Enhanced by IFITM Antiviral Activity

doi: 10.3389/fmicb.2022.840885

Figure Lengend Snippet: Arenavirus GPpp entry and MOPV replication is inhibited by IFN but is insensitive to IFITM overexpression. (A) A549 cells were infected with GFP-containing arenavirus GPpp in the presence or absence of type 1 interferon (IFN1) and infection was measured by flow cytometry. (B) A549 cells were infected with live MOPV (MOI = 0.01) in the presence or absence of IFN1. MOPV L or NP gene expression was determined by RT-qPCR of extracted RNA and infectious virus in cell supernatants was measured by plaque assay at indicated times post infection. (C) A549 cells were transduced with empty vector control pLHCX, IFITM1, IFITM2 or IFITM3. Expression of IFITMs and ZMPSTE24 was measured in the presence or absence of 1000 U/mL IFN1 by western blot analysis. HSP90 served as a loading control. A549 cells stably transduced with IFITMs were infected with arenavirus GPpp and% infectivity measured by flow cytometry (D) or were infected with live MOPV (MOI = 0.01) for 72 h and MOPV L or NP gene expression was measured by RT-qPCR of extracted RNA (E) . Data are shown as mean ± SE (standard error) of n = 3 independent experiments. Significance is indicated as p -values *** p < 0.0001, ** p < 0.001, * p < 0.05.

Article Snippet: Lysed samples were centrifuged and supernatants were immunoprecipitated with 5 μg/ml mouse monoclonal anti-IFITM2/3 antibody (Proteintech, 66081-1-Ig) for 1.5 h at 4°C.

Techniques: Over Expression, Infection, Flow Cytometry, Expressing, Quantitative RT-PCR, Plaque Assay, Transduction, Plasmid Preparation, Western Blot, Stable Transfection

Journal: Frontiers in Microbiology

Article Title: Inhibition of Arenavirus Entry and Replication by the Cell-Intrinsic Restriction Factor ZMPSTE24 Is Enhanced by IFITM Antiviral Activity

doi: 10.3389/fmicb.2022.840885

Figure Lengend Snippet: ZMPSTE24 colocalises with IFITM proteins and interacts via a C-terminal interaction with IFITM3. (A) ZMPSTE24 is localised to early and late endosomal compartments. ZMPSTE24-HA was transiently expressed in A549 cells which were then probed with antibodies against HA, early endosomal (EEA1) or late endosomal (Rab9) markers, or LAMP1. Nuclei were counterstained with DAPI. Panels are of representative images. Pearson’s correlation coefficient of ZMPSTE24 with the early and late endosomal compartment markers was calculated by measuring cells of interest using ImageJ software. (B) A549 cells transiently expressing HA-tagged IFITM proteins 1, 2, and 3 were probed with antibodies against endogenous ZMPSTE24 and HA and imaged by confocal microscopy. Panels are of representative images. Pearson’s correlation coefficient of ZMPSTE24 with individual IFITM proteins was calculated by measuring cells of interest using ImageJ software. (C) For the endogenous IP, A549 cells were pre-treated with IFN prior to cell lysis. For the co-IP, IFN pre-treated A549 cells were mock-transfected or transfected with ZMPSTE24-FLAG. Cell lysates were immunoprecipitated with anti-IFITM2/3 monoclonal antibody prior to resolving by SDS-PAGE and analysis by western blotting for endogenous ZMPSTE24 or ZMPSTE24-FLAG along with IFITMs 1, 2, and 3. (D) Schematic illustration of NanoBiT construct expression. IFITM3 or ZMPSTE24 were fused to large (LgBiT) or small (SmBiT) subunits of NanoLuc luciferase at N or C terminals and co-transfected in pairs (hatched boxes) into HEK293T cells. (E) Luminescence produced by interaction of co-expressed NanoBiT pairs was measured in live cells and analysed relative to a control consisting of the target-LgBiT co-transfected with a HaloTag-SmBiT control. LgBiT-PRKACA and SmBiT-PRKAR2A were co-transfected as a positive control. Green arrow indicates interaction between ZMPSTE24-C-SmBiT and IFITM3-C-LgBiT. Data are shown as mean ± SE (standard error) of n = 3 independent experiments. Significance is indicated as p -values *** p < 0.0001, ** p < 0.001.

Article Snippet: Lysed samples were centrifuged and supernatants were immunoprecipitated with 5 μg/ml mouse monoclonal anti-IFITM2/3 antibody (Proteintech, 66081-1-Ig) for 1.5 h at 4°C.

Techniques: Software, Expressing, Confocal Microscopy, Lysis, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, SDS Page, Western Blot, Construct, Luciferase, Produced, Positive Control