Journal: The EMBO journal

Article Title: Interferon-inducible phospholipids govern IFITM3-dependent endosomal antiviral immunity.

doi: 10.15252/embj.2022112234

Figure Lengend Snippet: Figure 7. IFITM1 and plasma membrane mutants IFITM3 inhibit SARS CoV2 entry and infection.

Article Snippet: Samples were subjected to SDS–PAGE using Bolt 4–12% Bis-Tris Plus Gels (Thermo Fisher Scientific), transferred to PVDF membrane by electroblotting, and blotted with rabbit anti-IFITM3 polyclonal Ab (1:1,000 dilution, proteintech Cat# 11714-1-AP); rabbit anti-IFITM2 polyclonal Ab (1:2,500, proteintech Cat# 12769-1-AP); mouse anti-IFITM1 monoclonal Ab (1:5,000, 2023 The Authors The EMBO Journal 42: e112234 | 2023 17 of 23 D ow nloaded from https://w w w .em bopress.org on January 13, 2024 from IP 180.252.95.111. proteintech Cat# 60074-1-Ig), rabbit anti-ACE2 (1:1,000, Cell Signaling Technology 4355S), rabbit anti-HA tag antibody—ChIP Grade (1:10,000 Abcam Cat# ab9110), mouse anti-his (1:10,000 Millipore Cat# 05-909).

Techniques: Clinical Proteomics, Membrane, Infection

Journal: Cell Reports

Article Title: Quantitative Temporal Proteomic Analysis of Vaccinia Virus Infection Reveals Regulation of Histone Deacetylases by an Interferon Antagonist

doi: 10.1016/j.celrep.2019.04.042

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used at the indicated dilution, in blocking solution: rabbit anti-IFIT1 (1:1000, cat. no. PA3-848, Thermo Fisher Scientific), mouse anti-IFITM1/2/3 (1:500, cat. no. sc-374026, Santa Cruz Biotechnology), mouse anti-COL6A2 (1:500, cat. no. sc-374566, Santa Cruz Biotechnology), mouse anti-COL1A2 (1:500, cat. no. sc-376350, Santa Cruz Biotechnology), mouse anti-TRIM5 (1:500, cat. no. SC-373864, Santa Cruz Biotechnology), mouse anti-HDAC5 (1:500, cat. no. sc-133225, Santa Cruz Biotechnology), mouse anti-HDAC1 (1:500, cat. no. sc-81598, Santa Cruz Biotechnology), mouse anti-FLAG, (1:1000, F3165, Sigma-Aldrich), mouse anti-Myc (1:1000, cat. no. 2276, Cell Signaling Technology), rat anti-α-tubulin (1:10,000, clone YL1/2, cat. no. MCA77G, Serotec), rabbit anti-C6 (1:1000, described in , mouse anti-D8 (1:1000, described in ( )).

Techniques: Virus, Western Blot, Derivative Assay, Subcloning, Recombinant, Protease Inhibitor, Electron Microscopy, Bicinchoninic Acid Protein Assay, TA Cloning, Sequencing, Mass Spectrometry, CRISPR, Disruption, Plasmid Preparation, Software, Quantitative Proteomics, Imaging, Flow Cytometry, Fluorescence, Microscopy

Journal: Intractable & Rare Diseases Research

Article Title: The novel role of IFITM1-3 in myogenic differentiation of C2C12 cells

doi: 10.5582/irdr.2023.01050

Figure Lengend Snippet: Primer sequences for RT-qPCR

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h at room temperature, and then the membranes were incubated with primary antibodies, namely, rabbit anti-desmin (ab32362, Abcam, USA), rabbit anti-MyoD (ab203383, Abcam, USA), rabbit anti-MYF5 (ab125301, Abcam, USA), rabbit anti-IFITM1 (bs-1031R, Bioss, China), rabbit anti-IFITM2 (bs-15517R, Bioss, China), mouse anti-IFITM3 (bsm-51629M, Bioss, China), and rabbit anti-GAPDH (ab8245, Abcam, USA), at 4°C overnight.

Techniques: Sequencing

Journal: Intractable & Rare Diseases Research

Article Title: The novel role of IFITM1-3 in myogenic differentiation of C2C12 cells

doi: 10.5582/irdr.2023.01050

Figure Lengend Snippet: Increased expression of IFITM1-3 during myogenic differentiation of C2C12 myoblasts. (A) Relative expression of Ifitm1-3 during the myogenic differentiation process. (B) Western blot evaluating the protein levels of IFITM1-3. (C) Quantification of band intensity as described above is shown. The level of proteins was normalized to that of GAPDH. Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h at room temperature, and then the membranes were incubated with primary antibodies, namely, rabbit anti-desmin (ab32362, Abcam, USA), rabbit anti-MyoD (ab203383, Abcam, USA), rabbit anti-MYF5 (ab125301, Abcam, USA), rabbit anti-IFITM1 (bs-1031R, Bioss, China), rabbit anti-IFITM2 (bs-15517R, Bioss, China), mouse anti-IFITM3 (bsm-51629M, Bioss, China), and rabbit anti-GAPDH (ab8245, Abcam, USA), at 4°C overnight.

Techniques: Expressing, Western Blot

Journal: Intractable & Rare Diseases Research

Article Title: The novel role of IFITM1-3 in myogenic differentiation of C2C12 cells

doi: 10.5582/irdr.2023.01050

Figure Lengend Snippet: Knockdown of Ifitm1, 2, and 3 by siRNAs blocks myogenic differentiation in C2C12 cells. (A) Microscopic images of Giemsa staining for C2C12 myoblasts on day 3 of myogenic differentiation after transfection with siRNAs targeting Ifitm1-3. Two different siRNAs were used for each targeting gene. Transfection without siRNAs, but with transfection reagent was set as mock. Magnification: 100×. (B) Percentage fusion on day 3 of myogenic induction and transfection with siRNAs as described above, calculated by dividing the number of nuclei within multinucleated myofibers by the total number of nuclei. NC represents the group without siRNAs and transfection reagents. (C) Downregulated protein expression of myogenin, MyoD, Myf5, and desmin after interference by siRNAs targeting Ifitm1-3. (D) Quantification of band intensity as described above is shown. The level of proteins was normalized to that of GAPDH. Statistical significance: *P< 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h at room temperature, and then the membranes were incubated with primary antibodies, namely, rabbit anti-desmin (ab32362, Abcam, USA), rabbit anti-MyoD (ab203383, Abcam, USA), rabbit anti-MYF5 (ab125301, Abcam, USA), rabbit anti-IFITM1 (bs-1031R, Bioss, China), rabbit anti-IFITM2 (bs-15517R, Bioss, China), mouse anti-IFITM3 (bsm-51629M, Bioss, China), and rabbit anti-GAPDH (ab8245, Abcam, USA), at 4°C overnight.

Techniques: Staining, Transfection, Expressing

Journal: Intractable & Rare Diseases Research

Article Title: The novel role of IFITM1-3 in myogenic differentiation of C2C12 cells

doi: 10.5582/irdr.2023.01050

Figure Lengend Snippet: Identification and validation of IFITM1,3-interacting proteins. (A) Principle of co-immunoprecipitation and liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). (B) The protein- protein interaction network if IFITM1,3 (overlapped) revealed by STRING analysis. A total of 84 unique homologous proteins are shown in the network. Three clusters are indicated in different colors. Cluster 1: muscle filament sliding (green color); Cluster 2: ribosome series proteins (red color); Cluster 3: regulation of mRNA metabolic process (blue color). Associations are represented by the lines. Thicker lines represent stronger associations. (C) Co-IP assays show the interaction between desmin and IFITM1, 3.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h at room temperature, and then the membranes were incubated with primary antibodies, namely, rabbit anti-desmin (ab32362, Abcam, USA), rabbit anti-MyoD (ab203383, Abcam, USA), rabbit anti-MYF5 (ab125301, Abcam, USA), rabbit anti-IFITM1 (bs-1031R, Bioss, China), rabbit anti-IFITM2 (bs-15517R, Bioss, China), mouse anti-IFITM3 (bsm-51629M, Bioss, China), and rabbit anti-GAPDH (ab8245, Abcam, USA), at 4°C overnight.

Techniques: Immunoprecipitation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay

Journal: Intractable & Rare Diseases Research

Article Title: The novel role of IFITM1-3 in myogenic differentiation of C2C12 cells

doi: 10.5582/irdr.2023.01050

Figure Lengend Snippet: Overlapped interacted proteins for IFITM1 and IFITM3

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h at room temperature, and then the membranes were incubated with primary antibodies, namely, rabbit anti-desmin (ab32362, Abcam, USA), rabbit anti-MyoD (ab203383, Abcam, USA), rabbit anti-MYF5 (ab125301, Abcam, USA), rabbit anti-IFITM1 (bs-1031R, Bioss, China), rabbit anti-IFITM2 (bs-15517R, Bioss, China), mouse anti-IFITM3 (bsm-51629M, Bioss, China), and rabbit anti-GAPDH (ab8245, Abcam, USA), at 4°C overnight.

Techniques:

Journal: Intractable & Rare Diseases Research

Article Title: The novel role of IFITM1-3 in myogenic differentiation of C2C12 cells

doi: 10.5582/irdr.2023.01050

Figure Lengend Snippet: GO and KEGG pathway enrichment analysis of 84 proteins that interact with IFITM1, 3 (overlapped). (A) KEGG classification map of differentially expressed genes. The y-axis shows the metabolic pathway. (B) Biological process (BP). (C) Cellular component (CC). (D) Molecular function (MF). The x-axis represents gene ratio = count/set size. Dot size represents the number of genes, and the color bar represents the Padj-value.

Article Snippet: The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h at room temperature, and then the membranes were incubated with primary antibodies, namely, rabbit anti-desmin (ab32362, Abcam, USA), rabbit anti-MyoD (ab203383, Abcam, USA), rabbit anti-MYF5 (ab125301, Abcam, USA), rabbit anti-IFITM1 (bs-1031R, Bioss, China), rabbit anti-IFITM2 (bs-15517R, Bioss, China), mouse anti-IFITM3 (bsm-51629M, Bioss, China), and rabbit anti-GAPDH (ab8245, Abcam, USA), at 4°C overnight.

Techniques:

Journal: The Journal of Infectious Diseases

Article Title: Interferon-Induced Transmembrane Protein–Mediated Inhibition of Host Cell Entry of Ebolaviruses

doi: 10.1093/infdis/jiv255

Figure Lengend Snippet: Interferon-induced transmembrane (IFITM) proteins inhibit entry driven by glycoproteins representing all viral species within the genus Ebolavirus. A , 293T cells, transduced to express human IFITM1, IFITM2, IFITM3, or chloramphenicol acetyltransferase (Cat), were subsequently transduced with vectors encoding luciferase and bearing the indicated viral entry proteins. Means are shown for 3 independent experiments performed with triplicate samples. Transduction of Cat-expressing cells (control) was set as 100%. Error bars indicate standard errors of the mean (SEMs). B , Experiment conducted as described in A but with examination of cells expressing human ( top ) or rhesus macaque ( bottom ) IFITM proteins. Means and SEMs are shown for 3 independent experiments performed with triplicate samples. Transduction of Cat-expressing cells (control) was set as 100%. C , Expression of myc-tagged IFITM proteins in transduced 293 T cells was analyzed by Western blot using anti-myc antibody. D , Expression of IFITM-cyan fluorescent protein fusion proteins in transduced 293T cells was analyzed by flow cytometry. Single representative experiments are shown in C and D and were confirmed in ≥1 separate experiment. Abbreviations: BDBV, Bundibugyo virus; EBOV, Ebola virus; FLUAV, influenza A virus; GP, glycoprotein; HA, hemagglutinin; LASV, Lassa virus; MACV, Machupo virus; MARV, Marburg virus; MLV, murine leukemia virus; RESTV, Reston virus; SUDV, Sudan virus; TAFV, Taï Forest virus; VSV, vesicular stomatitis virus.

Article Snippet: Expression of IFITM proteins was detected using Western blot analyses with a monoclonal mouse anti-IFITM1 antibody (1:1000, Proteintech) and polyclonal rabbit anti-IFITM 2 antibody (1:2000, Proteintech), which is cross-reactive against IFITM3.

Techniques: Transduction, Luciferase, Expressing, Control, Western Blot, Flow Cytometry, Virus

Journal: The Journal of Infectious Diseases

Article Title: Interferon-Induced Transmembrane Protein–Mediated Inhibition of Host Cell Entry of Ebolaviruses

doi: 10.1093/infdis/jiv255

Figure Lengend Snippet: Stimulation with type I interferon (IFN) induces IFN-induced transmembrane (IFITM) protein expression in human macrophages. Monocyte-derived macrophages were incubated with the indicated IFNs for 24 hours or left untreated and expression of STAT1, phosphorylated STAT1, β-actin, IFITM1, and IFITM2/3 was assessed by Western blot analysis. As controls, 293T cells were transfected with empty plasmid or expression plasmids for IFITM1–3. Similar results were obtained in 3 separate experiments. Abbreviation: MDM, monocyte-derived macrophages.

Article Snippet: Expression of IFITM proteins was detected using Western blot analyses with a monoclonal mouse anti-IFITM1 antibody (1:1000, Proteintech) and polyclonal rabbit anti-IFITM 2 antibody (1:2000, Proteintech), which is cross-reactive against IFITM3.

Techniques: Expressing, Derivative Assay, Incubation, Western Blot, Transfection, Plasmid Preparation

Journal: The Journal of Infectious Diseases

Article Title: Interferon-Induced Transmembrane Protein–Mediated Inhibition of Host Cell Entry of Ebolaviruses

doi: 10.1093/infdis/jiv255

Figure Lengend Snippet: Amphotericin B does not rescue Ebola virus (EBOV) glycoprotein (GP)–driven entry from inhibition by interferon-induced transmembrane (IFITM) proteins. After 293T cells were transduced to express IFITM1, IFITM2, and IFITM3 or chloramphenicol acetyltransferase (control) and treated with 10 µmol/L amphotericin B for 1 hour, they were then transduced with luciferase-encoding murine leukemia virus particles bearing EBOV-GP or influenza A virus (FLUAV) hemagglutinin (HA). Means are shown for 3 independent experiments; error bars represent standard errors of the mean. Transduction of control cells was set as 100%.

Article Snippet: Expression of IFITM proteins was detected using Western blot analyses with a monoclonal mouse anti-IFITM1 antibody (1:1000, Proteintech) and polyclonal rabbit anti-IFITM 2 antibody (1:2000, Proteintech), which is cross-reactive against IFITM3.

Techniques: Virus, Inhibition, Control, Transduction, Luciferase

Journal: The Journal of Infectious Diseases

Article Title: Interferon-Induced Transmembrane Protein–Mediated Inhibition of Host Cell Entry of Ebolaviruses

doi: 10.1093/infdis/jiv255

Figure Lengend Snippet: Inhibition of Lloviu virus (LLOV) glycoprotein (GP)–driven entry by interferon-induced transmembrane (IFITM) proteins. A , 293T cells transduced to express human IFITM1, 2, 3 or chloramphenicol acetyltransferase (Cat) as a control were subsequently transduced with vectors bearing the indicated GPs and luciferase activity in cell lysates was determined. Means and standard errors of the mean (SEMs) are shown for 3 independent experiments performed with triplicate samples. Transduction of control cells was set as 100%. Abbreviations: FLUAV-HA, influenza A virus hemagglutinin; MLV-Env, murine leukemia virus . B , RD/ Gaussia luciferase (GLucC) cells transduced to express human IFITM 3 or Cat as control were spin-infected with VP40/N-terminal portion of GLuc virus-like particles (VLPs) bearing the indicated GPs, and the luciferase activity in cell lysates was determined. Means and SEMs are shown for 2 experiments conducted with quintuplicate samples. Infection of control cells was set as 100%. Similar results were obtained when 293T cells transfected to express IFITM3 were analyzed. C , Western blot analysis of IFITM expression in transduced 293T cells. The expression of wild type-IFITM3 and an IFITM3 mutant in which the amino acids SVKS were mutated to alanine was analyzed. D , Experiment was conducted as described for A , but the SVKS-AAAA mutant was analyzed. Means and SEMs are shown for 3 independent experiments. Transduction of control cells was set as 100%. Abbreviation: MARV, Marburg virus. E , Experiment was carried as described for B, but the IFITM3 SVKS-AAAA mutant was included, and VLPs bearing no GP served as negative control. Results of a single experiment conducted with quintuplicate samples are shown and were confirmed in a separate experiment; error bars indicate standard deviations.

Article Snippet: Expression of IFITM proteins was detected using Western blot analyses with a monoclonal mouse anti-IFITM1 antibody (1:1000, Proteintech) and polyclonal rabbit anti-IFITM 2 antibody (1:2000, Proteintech), which is cross-reactive against IFITM3.

Techniques: Inhibition, Virus, Control, Transduction, Luciferase, Activity Assay, Infection, Transfection, Western Blot, Expressing, Mutagenesis, Negative Control

Journal: The Journal of Infectious Diseases

Article Title: Interferon-Induced Transmembrane Protein–Mediated Inhibition of Host Cell Entry of Ebolaviruses

doi: 10.1093/infdis/jiv255

Figure Lengend Snippet: Synergistic inhibition of Ebola virus (EBOV) glycoprotein (GP)–driven entry by interferon-induced transmembrane (IFITM) proteins and GP-specific antiserum. 293T cells transduced to express IFITM1, IFITM2, and IFITM3 or chloramphenicol acetyltransferase as a control were transduced with vectors bearing the indicated GPs, which were preincubated with medium alone (control), 1:500 diluted rabbit preimmune serum, or 1:500 diluted rabbit anti–EBOV-GP antiserum for 30 minutes. The means and standard errors of the mean for 2 (Lassa virus [LASV]–GPC) or 4 (EBOV-GP and influenza A virus [FLUAV] hemagglutinin [HA]) experiments are shown.

Article Snippet: Expression of IFITM proteins was detected using Western blot analyses with a monoclonal mouse anti-IFITM1 antibody (1:1000, Proteintech) and polyclonal rabbit anti-IFITM 2 antibody (1:2000, Proteintech), which is cross-reactive against IFITM3.

Techniques: Inhibition, Virus, Control, Transduction

Journal: Oncogenesis

Article Title: A mesenchymal glioma stem cell profile is related to clinical outcome

doi: 10.1038/oncsis.2014.5

Figure Lengend Snippet: Hierarchical clustering analysis of GSCs. ( a ) The unsupervised clustering using Ward's linkage method and Euclidean distance individualized H9 HNSCs, H1-H9-ESCs and two groups of GSCs (GSCX-1 and GSCX-2). Note that culture conditions of H9 HNSC do not influence the expression of pluripotency gene samples H9 HNSC-1 and H9 HNSC-2 samples. ( b ) Box plot representation of COL1A1 and IFITM1 expression levels between GSC groups GSCX-1 and GSCX-2. The charts show the log 10 expression of relative quantification (RQ) values normalized to the expression of H9 HNSC cell line. ( c ) Box plot representation of COL1A1 and IFITM1 expression levels between bulk tumor groups TX-1 and TX-2. The top edge of the boxes represents the 75th percentile, the bottom edge, the 25th percentile and × the mean. The range is shown as vertical edge.

Article Snippet: Antibodies used in this study were goat anti-IFITM1 antibody (R&D Systems Europe, Lille, France), rabbit anti-COL1A1 antibody (Abcam, Paris, France) and mouse β-actin antibody (Abcam).

Techniques: Expressing, Quantitative Proteomics

Journal: Oncogenesis

Article Title: A mesenchymal glioma stem cell profile is related to clinical outcome

doi: 10.1038/oncsis.2014.5

Figure Lengend Snippet: Knockdown efficiency of COL1A1 and IFITM1 and effect on cell invasion. ( a ) COL1A1 and IFITM1 mRNA expression was monitored by quantitative reverse transcriptase–PCR in GSC-3 and GSC-9 ( n =2). Significant inhibition of 90% of mRNA expression was observed. GADPH was used as internal control. * P <0.05. Each value is expressed as mean±s.e.m. ( b ) Western blot analysis showed diminution of IFITM1 and COL1A1 proteins expression, β-actin was used as loading control. ( c ) Densitometry of protein level. The bar graph shows relative IFITM1 and COL1A1 protein expression levels expressed as percentage of IFITM1 and COL1A1 expression in control cells ( n =3). We observed >80% inhibition for both genes in GSC-3 and GSC-9 cells, as compared with control cells. ( d ) Representative fields of Boyden cell invasion assay. After 3 days of invasion, cells that migrated across the membrane were fixed and stained with hematoxylin/eosin and were counted in one hundred different fields. The invaded cells were photographed under the microscope at × 100 magnification. ( e ) A significant reduction of invasion is observed in both GSC-3 and GSC-9 cells lines with sh-IFITM1 as well as for sh-COL1A1. The results represent a percentage of invaded cells. Data are presented as mean±s.e.m. from three independent experiments. * P <0.05, ** P <0.01.

Article Snippet: Antibodies used in this study were goat anti-IFITM1 antibody (R&D Systems Europe, Lille, France), rabbit anti-COL1A1 antibody (Abcam, Paris, France) and mouse β-actin antibody (Abcam).

Techniques: Knockdown, Expressing, Reverse Transcription, Inhibition, Control, Western Blot, Invasion Assay, Membrane, Staining, Microscopy

Journal: Oncogenesis

Article Title: A mesenchymal glioma stem cell profile is related to clinical outcome

doi: 10.1038/oncsis.2014.5

Figure Lengend Snippet: Effects of COL1A1 and IFITM1 inhibition on cell proliferation and on neurosphere-initiating capacity. ( a ) Cell proliferation was measured by using a MTS assay. Each experiment was carried out in replicates of six and repeated twice and expressed as mean±s.d. ( b ) Inhibition of COL1A1 resulted in a significant (* P <0.05) decrease of neurosphere-initiating cell (NS-IC) frequency whereas inhibition of IFITM1 expression only slightly modified the neurosphere-initiating capacity. Final cell dilutions yielding 37% of negative wells correspond to the dilution at which there is one NS-ICs per well. Each experiment was performed in duplicate and repeated twice and expressed as mean±s.d.

Article Snippet: Antibodies used in this study were goat anti-IFITM1 antibody (R&D Systems Europe, Lille, France), rabbit anti-COL1A1 antibody (Abcam, Paris, France) and mouse β-actin antibody (Abcam).

Techniques: Inhibition, MTS Assay, Expressing, Modification

Journal: Oncogenesis

Article Title: A mesenchymal glioma stem cell profile is related to clinical outcome

doi: 10.1038/oncsis.2014.5

Figure Lengend Snippet: COL1A1 and IFITM1 expression levels in an independent cohort of 30 patients' tumors. ( a ) Box plot representation of the mRNA expression levels. The charts show the log 10 expression of relative quantification (RQ) values normalized to the expression of H9 NSC cell line. The top edge of the boxes represents the 75th percentile, the bottom edge, the 25th percentile and × the mean. The range is shown as vertical edge. ( b ) Kaplan–Meier analysis for overall survival of an independent cohort of 30 GBM patients. Survival distribution of patients with low (group A; n =13) versus high (group B; n =17) expression of COL1A1 and IFITM1 ( P <10 −4 ).

Article Snippet: Antibodies used in this study were goat anti-IFITM1 antibody (R&D Systems Europe, Lille, France), rabbit anti-COL1A1 antibody (Abcam, Paris, France) and mouse β-actin antibody (Abcam).

Techniques: Expressing, Quantitative Proteomics

Journal: Viruses

Article Title: Antiviral Role of IFITM Proteins in Classical Swine Fever Virus Infection

doi: 10.3390/v11020126

Figure Lengend Snippet: Primers used in this study.

Article Snippet: Following blocking in 5% non-fat milk, the membranes were incubated with antibodies including mouse anti-Flag monoclonal antibody (MAb; CWBIO), rabbit anti-IFITM1 polyclonal antibody (PAb; Cusabio, Wuhan, China), rabbit anti-IFITM2/3 PAb (Cusabio), mouse anti-GFP MAb (SUNGENE BIOTECH, Tianjin, China), and mouse anti-β-actin MAb (CWBIO) at 4 °C overnight.

Techniques: Sequencing, Amplification, Negative Control, Knockdown

Journal: Viruses

Article Title: Antiviral Role of IFITM Proteins in Classical Swine Fever Virus Infection

doi: 10.3390/v11020126

Figure Lengend Snippet: Overexpression of IFITMs inhibits classical swine fever virus (CSFV) replication in porcine alveolar macrophages (PAMs). ( A ) Cell viability of cell lines stably overexpressing IFITMs. ( B ) Confirmation of CMV-IFITM1, CMV-IFITM2, and CMV-IFITM3 lentivirus transfection by detection of enhanced green fluorescent protein (EGFP) reporter. ( a ) Mock-transfected PAMs. ( b ) PAMs transfected with lentiviruses expressing CMV. ( c ) PAMs transfected with CMV-IFITM1 lentivirus, CMV-IFITM2 lentivirus ( d ), and CMV-IFITM3 lentivirus ( e ). Scale bars, 100 μm. ( C ) Western blot analysis of PAM cell lines stably expressing Flag-tagged IFITM1, IFITM2, or IFITM3 or CMV alone using an anti-Flag antibody. ( D ) Western blot analysis of IFITMs in PAM cell lines stably expressing Flag-tagged IFITM1, IFITM2, or IFITM3 or CMV alone using an IFITM1-specific antibody and an IFITM2/3 antibody against IFITM2 and IFITM3, respectively. β-actin served as an internal control. ( E ) CSFV genomic RNA in CMV-IFITM1, CMV-IFITM2, and CMV-IFITM3 cell lines. The CMV, CMV-IFITM1, CMV-IFITM2, and CMV-IFITM3 cell lines were infected with CSFV at a multiplicity of infection (MOI) of 1. CSFV genomic RNA levels were determined by real-time quantitative PCR (RT-qPCR) at 12 and 24 h post-infection (hpi). Data were normalized to β-actin expression. ( F ) Infectious progeny viral titers in culture medium from CMV-IFITM1, CMV-IFITM2, and CMV-IFITM3 cells. The viral titers of CSFV in supernatants were quantified by an immunofluorescence assay (IFA) and expressed as tissue culture infective dose (TCID 50 )/mL. Data ( A , E , F ) represent the mean ± SD of three independent experiments and were measured in technical duplicate. Comparisons between groups were performed by Student’s t test. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Following blocking in 5% non-fat milk, the membranes were incubated with antibodies including mouse anti-Flag monoclonal antibody (MAb; CWBIO), rabbit anti-IFITM1 polyclonal antibody (PAb; Cusabio, Wuhan, China), rabbit anti-IFITM2/3 PAb (Cusabio), mouse anti-GFP MAb (SUNGENE BIOTECH, Tianjin, China), and mouse anti-β-actin MAb (CWBIO) at 4 °C overnight.

Techniques: Over Expression, Virus, Stable Transfection, Transfection, Expressing, Western Blot, Control, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence

Journal: Viruses

Article Title: Antiviral Role of IFITM Proteins in Classical Swine Fever Virus Infection

doi: 10.3390/v11020126

Figure Lengend Snippet: Knockdown of IFITMs mediated by short hairpin RNA (shRNA) enhances CSFV replication. ( A ) Cell viability of stable IFITM-knockdown cell line. ( B ) Confirmation of recombinant IFITM shRNA lentivirus (shIFITM) transfection by detection of EGFP reporter. ( a ) Mock-transfected PAMs. ( b ) PAMs transfected with scrambled shRNA lentivirus (shN). ( c ) PAMs transfected with shIFITM. Scale bars, 100 μm. ( C ) Western blot analysis of IFITM expression in IFITM-knockdown cells using an IFITM1-specific antibody and an IFITM2/3 antibody against IFITM2 and IFITM3, respectively. β-actin served as an internal control. ( D ) Western blot analysis of IFITM expression using an anti-Flag antibody in shIFITM cell lines following transfection with CMV-IFITM1, CMV-IFITM2, or CMV-IFITM3 for 48 h. β-actin served as an internal control. ( E ) CSFV genomic RNA in shIFITM cell lines. The shN and shIFITM cell lines were infected with CSFV at an MOI of 1. CSFV genomic RNA levels were determined by RT-qPCR at 12 and 24 hpi. Data were normalized to β-actin expression. ( F ) Infectious progeny viral titers in culture medium from shIFITM cells. The viral titers of CSFV in supernatants were quantified and expressed as TCID 50 /mL. Data (A, E, and F) represent the mean ± SD of three independent experiments and were measured in technical duplicate. Comparisons between groups were performed by Student’s t test. * p < 0.05 and *** p < 0.001.

Article Snippet: Following blocking in 5% non-fat milk, the membranes were incubated with antibodies including mouse anti-Flag monoclonal antibody (MAb; CWBIO), rabbit anti-IFITM1 polyclonal antibody (PAb; Cusabio, Wuhan, China), rabbit anti-IFITM2/3 PAb (Cusabio), mouse anti-GFP MAb (SUNGENE BIOTECH, Tianjin, China), and mouse anti-β-actin MAb (CWBIO) at 4 °C overnight.

Techniques: Knockdown, shRNA, Recombinant, Transfection, Western Blot, Expressing, Control, Infection, Quantitative RT-PCR

Journal: Viruses

Article Title: Antiviral Role of IFITM Proteins in Classical Swine Fever Virus Infection

doi: 10.3390/v11020126

Figure Lengend Snippet: Expression of IFITMs is induced by interferon (IFN)-α treatment. RT-qPCR analysis of IFITM1 ( A ) and IFITM2/3 ( B ) mRNA levels (normalized to β-actin expression) in PAMs treated with IFN-α (10 ng/mL) at 6, 12, 18, and 24 h post-treatment (hpt). ( C ) Western blot analysis of IFITM expression in PAMs treated with IFN-α (10 ng/mL) at 6, 12, 18, and 24 hpt. β-actin served as an internal control. ( D ) CSFV genomic RNA in shIFITM cell lines with IFN-α pre-treatment. The shN and shIFITM cell lines were pre-treated with IFN-α and then infected with CSFV at an MOI of 1. CSFV genomic RNA levels were determined by RT-qPCR at 24 hpi. Data were normalized to β-actin expression. RT-qPCR analysis of IFITM1 ( E ), IFITM2/3 ( F ) and Mx1 ( G ) mRNA levels (normalized to β-actin expression) in shN and shIFITM cells. Data ( A , B , D , E – G ) represent the mean ± SD of three independent experiments and were measured in technical duplicate. Comparisons between groups were performed by Student’s t test. ** p < 0.01 and *** p < 0.001

Article Snippet: Following blocking in 5% non-fat milk, the membranes were incubated with antibodies including mouse anti-Flag monoclonal antibody (MAb; CWBIO), rabbit anti-IFITM1 polyclonal antibody (PAb; Cusabio, Wuhan, China), rabbit anti-IFITM2/3 PAb (Cusabio), mouse anti-GFP MAb (SUNGENE BIOTECH, Tianjin, China), and mouse anti-β-actin MAb (CWBIO) at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Infection

Journal: Viruses

Article Title: Antiviral Role of IFITM Proteins in Classical Swine Fever Virus Infection

doi: 10.3390/v11020126

Figure Lengend Snippet: IFITM expression is downregulated by CSFV. RT-qPCR analysis of IFITM1 ( A ) and IFITM2/3 ( B ) mRNA levels (normalized to β-actin expression) in PAMs infected with CSFV at an MOI of 0.1, 1, or 3 for 24 h. Data represent the mean ± SD of three independent experiments and were measured in technical duplicate. Comparisons between groups were performed by Student’s t test. * p < 0.05 and *** p < 0.001 ( C ) Western blot analysis of IFITM expression in PAMs infected with different doses of CSFV (MOI = 0.1, 1, or 3) for 24 h. β-actin served as an internal control.

Article Snippet: Following blocking in 5% non-fat milk, the membranes were incubated with antibodies including mouse anti-Flag monoclonal antibody (MAb; CWBIO), rabbit anti-IFITM1 polyclonal antibody (PAb; Cusabio, Wuhan, China), rabbit anti-IFITM2/3 PAb (Cusabio), mouse anti-GFP MAb (SUNGENE BIOTECH, Tianjin, China), and mouse anti-β-actin MAb (CWBIO) at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR, Infection, Western Blot, Control

Journal: Viruses

Article Title: Antiviral Role of IFITM Proteins in Classical Swine Fever Virus Infection

doi: 10.3390/v11020126

Figure Lengend Snippet: Distribution of IFITMs in CSFV-infected PAMs. ( A ) Confirmation of C1-IFITM1, C1-IFITM2, and C1-IFITM3 transfection in PAMs by western blot detection of fusion proteins with an anti-GFP antibody. PAMs were transfected with C1-IFITM1, C1-IFITM2, C1-IFITM3, or C1-EGFP for 48 h and collected for western blot analysis. β-actin served as an internal control. ( B ) Confocal images of PAMs expressing EGFP-tagged IFITM1, 2, or 3 or EGFP alone. PAMs were transfected with C1-EGFP ( a ), C1-IFITM1 ( b ), C1-IFITM2 ( c ), or C1-IFITM3 ( d ) for 24 h, followed by mock infection or infection with CSFV (MOI = 1) for another 24 h. Cells were fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bars, 10 μm.

Article Snippet: Following blocking in 5% non-fat milk, the membranes were incubated with antibodies including mouse anti-Flag monoclonal antibody (MAb; CWBIO), rabbit anti-IFITM1 polyclonal antibody (PAb; Cusabio, Wuhan, China), rabbit anti-IFITM2/3 PAb (Cusabio), mouse anti-GFP MAb (SUNGENE BIOTECH, Tianjin, China), and mouse anti-β-actin MAb (CWBIO) at 4 °C overnight.

Techniques: Infection, Transfection, Western Blot, Control, Expressing, Staining