Journal: Computational and Mathematical Methods in Medicine

Article Title: miR-152-3p Affects the Progression of Colon Cancer via the KLF4/IFITM3 Axis

doi: 10.1155/2020/8209504

Figure Lengend Snippet: The overexpression of KLF4 mediates the IFITM3 expression to regulate colon cancer cell proliferation and apoptosis. (a) The expression of KLF4 and IFITM3 in the normal cell line CCD-18Co and colon cell lines HT29, HCT116, and SW480 was detected by qRT-PCR. (b) Western blot was carried out for the protein examination of KLF4 and IFITM3 in cells transfected with oe-NC+pre-NC, oe-KLF4+pre-NC, and oe-KLF4+pre-IFITM3. (c) CCK-8, (d) colony formation assay, and (e) flow cytometry were performed to determine cell viability, colony-forming ability, and cell apoptosis (∗ means p < 0.05).

Article Snippet: After being blocked in 5% skim milk at room temperature for 1 h, the membranes were incubated overnight at 4°C with primary antibodies, followed by horseradish peroxidase- (HRP-) labeled secondary antibody goat anti-rabbit IgG H&L (ab6721, 1 : 2000, Abcam, Cambridge, UK) at room temperature for 1 h. Primary antibodies included KLF4 rabbit polyclonal antibody (ab215036, 1 : 1000, Abcam, Cambridge, UK), IFITM3 rabbit polyclonal antibody (ab109429, 1 : 1000, Abcam, Cambridge, UK), and GAPDH rabbit polyclonal antibody (ab9485, 1 : 2500, Abcam, Cambridge, UK).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Transfection, CCK-8 Assay, Colony Assay, Flow Cytometry

Journal: Computational and Mathematical Methods in Medicine

Article Title: miR-152-3p Affects the Progression of Colon Cancer via the KLF4/IFITM3 Axis

doi: 10.1155/2020/8209504

Figure Lengend Snippet: miR-152-3p targets KLF4 and decreases its expression. (a) Binding sites of miR-152-3p on KLF4 3′UTR were predicted by the bioinformatics method. (b) Dual-luciferase assay was done for the validation of the targeting relationship between miR-152-3p and KLF4. (c) Western blot was conducted to test the protein expression of KLF4 and IFITM3 in each treatment group (∗ means p < 0.05).

Article Snippet: After being blocked in 5% skim milk at room temperature for 1 h, the membranes were incubated overnight at 4°C with primary antibodies, followed by horseradish peroxidase- (HRP-) labeled secondary antibody goat anti-rabbit IgG H&L (ab6721, 1 : 2000, Abcam, Cambridge, UK) at room temperature for 1 h. Primary antibodies included KLF4 rabbit polyclonal antibody (ab215036, 1 : 1000, Abcam, Cambridge, UK), IFITM3 rabbit polyclonal antibody (ab109429, 1 : 1000, Abcam, Cambridge, UK), and GAPDH rabbit polyclonal antibody (ab9485, 1 : 2500, Abcam, Cambridge, UK).

Techniques: Expressing, Binding Assay, Luciferase, Western Blot

Journal: Computational and Mathematical Methods in Medicine

Article Title: miR-152-3p Affects the Progression of Colon Cancer via the KLF4/IFITM3 Axis

doi: 10.1155/2020/8209504

Figure Lengend Snippet: miR-152-3p affects colon cancer cell proliferation and apoptosis via the KLF4/IFITM3 axis. (a) Western blot was carried out to determine the protein expression of KLF4 and IFITM3 in cells transfected with inhibitor NC+si-NC, miR-152-3p inhibitor+si-NC, and miR-152-3p inhibitor+si-KLF4. (b) CCK-8, (c) colony formation assay, and (d) flow cytometry were conducted to assay cell viability, colony-forming ability, and apoptosis in each treatment group (∗ means p < 0.05).

Article Snippet: After being blocked in 5% skim milk at room temperature for 1 h, the membranes were incubated overnight at 4°C with primary antibodies, followed by horseradish peroxidase- (HRP-) labeled secondary antibody goat anti-rabbit IgG H&L (ab6721, 1 : 2000, Abcam, Cambridge, UK) at room temperature for 1 h. Primary antibodies included KLF4 rabbit polyclonal antibody (ab215036, 1 : 1000, Abcam, Cambridge, UK), IFITM3 rabbit polyclonal antibody (ab109429, 1 : 1000, Abcam, Cambridge, UK), and GAPDH rabbit polyclonal antibody (ab9485, 1 : 2500, Abcam, Cambridge, UK).

Techniques: Western Blot, Expressing, Transfection, CCK-8 Assay, Colony Assay, Flow Cytometry

Journal: Journal of Virology

Article Title: The Interferon-Stimulated Gene IFITM3 Restricts Infection and Pathogenesis of Arthritogenic and Encephalitic Alphaviruses

doi: 10.1128/jvi.00655-16

Figure Lengend Snippet: Figure 1. CHIKV infection is enhanced in cells lacking Ifitm3 expression. WT, 502

Article Snippet: Polyclonal rabbit anti-Ifitm3 235 (Proteintech, 11714-1-AP) was used for Ifitm3 detection in these experiments.

Techniques: Infection, Expressing

Journal: Journal of Virology

Article Title: The Interferon-Stimulated Gene IFITM3 Restricts Infection and Pathogenesis of Arthritogenic and Encephalitic Alphaviruses

doi: 10.1128/jvi.00655-16

Figure Lengend Snippet: Figure 4. Role of Ifitm3 in restricting CHIKV binding, entry and pH dependent 549

Article Snippet: Polyclonal rabbit anti-Ifitm3 235 (Proteintech, 11714-1-AP) was used for Ifitm3 detection in these experiments.

Techniques: Binding Assay

Journal: Journal of Virology

Article Title: The Interferon-Stimulated Gene IFITM3 Restricts Infection and Pathogenesis of Arthritogenic and Encephalitic Alphaviruses

doi: 10.1128/jvi.00655-16

Figure Lengend Snippet: Figure 5. Ifitm3 restricts CHIKV pathogenesis in vivo. Four week-old WT and Ifitm3-/- 566

Article Snippet: Polyclonal rabbit anti-Ifitm3 235 (Proteintech, 11714-1-AP) was used for Ifitm3 detection in these experiments.

Techniques: In Vivo

Journal: Journal of Virology

Article Title: The Interferon-Stimulated Gene IFITM3 Restricts Infection and Pathogenesis of Arthritogenic and Encephalitic Alphaviruses

doi: 10.1128/jvi.00655-16

Figure Lengend Snippet: Figure 6. Infection of splenocyte subsets by CHIKV-LR in WT and Ifitm3-/- mice. 582

Article Snippet: Polyclonal rabbit anti-Ifitm3 235 (Proteintech, 11714-1-AP) was used for Ifitm3 detection in these experiments.

Techniques: Infection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Δ20 IFITM2 differentially restricts X4 and R5 HIV-1

doi: 10.1073/pnas.1619640114

Figure Lengend Snippet: Characterization of Δ20 IFITM2. (A) Jurkat E6-1 or LCL cells carrying IFITM3 polymorphism rs12252-C/C, rs12252-T/C, or rs12252-T/T were treated with 1,000 units (U)/mL IFN-β. Two days later, cells were analyzed by quantitative RT-PCR (qRT-PCR) using specific primers for the indicated IFITM cDNA. DNA electrophoresis was performed to show that a specific Δ20 IFITM2, but not Δ21 IFITM3, transcript could be detected. PCR products of synthetic IFITM cDNA served as positive controls. (B) Schematic representation of three major IFITM2 transcripts. RNA expression of these transcripts in whole blood cells analyzed by RNA sequencing was adapted from ref. 21. RPKM, reads per kilobase per million mapped reads. Expression of IFITM mRNA transcripts in anti-CD3 and anti-CD28 antibody-activated CD4+ T cells (C) or moDCs (D) was analyzed by qRT-PCR. Expression of Δ20 IFITM2 transcript ENST00000602569 is shown. Error bars denote 1 SD (n = 3) (IFITM2 primers used for qRT-PCR are shown in Fig. S1A). (E) Anti-CD3 and anti-CD28 antibody-activated CD4+ T cells were treated with or without 1,000 U/mL IFN-β. Two days later, expression of IFITM proteins was analyzed by Western blotting using the indicated antibodies (details of these antibodies are shown in Fig. S2 A and B and Table S1). Protein bands from Jurkat E6-1 cells stably expressing the indicated IFITM proteins were used as size markers. Δ20 IFITM2 translates of both IFITM2 rs1059091-A and rs1059091-G polymorphisms, which have different migration, were included. The rs1059091-G Δ20 IFITM2 was used for our subsequent hyperexpression experiments. Δ20 IFITM2 indicates rs1059091-G Δ20 IFITM2 if there is no additional specification. Vector-transduced Jurkat E6-1 cells or Jurkat E6-1 cells expressing Δ20 IFITM2 (F) or FL IFITM2 (G) were labeled with an anti-IFITM2/3/Δ20 antibody and 4′,6-diamidino-2-phenylindole (DAPI) and imaged by confocal microscopy.

Article Snippet: Note that the upper bands detected by the anti-IFITM2/3/Δ20 antibody could be FL IFITM2 or IFITM3. ( H ) Experiments were similar to the experiments in C except that anti-CD3 and anti-CD28 antibody-activated CD4 + T cells were treated with media, 1,000 U/mL IFN-β, or 1 μg/mL PHA for 2 d. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Providers Immunogens Protein recognized Anti-IFITM1 (goat) R&D Systems (catalog no. AF4827) IFITM1 Met1-His36 IFITM1 Anti-IFITM2 (mouse) Proteintech (catalog no. 66137-1-Ig) IFITM2 FL FL IFITM2 Anti-IFITM2/3 (goat) R&D Systems (catalog no. AF4834) IFITM3 His3-His57 FL IFITM2, IFITM3, Δ20 IFITM2 (weakly) Anti-IFITM2/3/Δ20 (rabbit) Cell Signaling Technology (catalog no. 13530) IFITM2 around pro41 FL IFITM2, IFITM3, Δ20 IFITM2 Anti-IFITM1/2/3 (rabbit) ProSci (catalog no. 5807) IFITM1 FL IFITM1, FL IFITM2, IFITM3, Δ20 IFITM2 Open in a separate window Properties of anti-IFITM antibodies used in our studies Like other IFITM proteins, Δ20 IFITM2 expression was up-regulated by IFNs in CD4 + T cells, moDCs, and macrophages, whereas phytohemagglutinin (PHA), which has been widely used for propagating HIV-1, depleted its expression ( and and ).

Techniques: Quantitative RT-PCR, Nucleic Acid Electrophoresis, RNA Expression, RNA Sequencing, Expressing, Western Blot, Stable Transfection, Migration, Plasmid Preparation, Labeling, Confocal Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Δ20 IFITM2 differentially restricts X4 and R5 HIV-1

doi: 10.1073/pnas.1619640114

Figure Lengend Snippet: Characterization of expression of Δ20 IFITM2. (A) Schematic representation of amino acid sequences of IFITM1, IFITM2, IFITM3, and Δ20 IFITM2. Identical amino acid sequences among different IFITM proteins are colored in red. Immunogens of commercial antibodies used in our studies are shown. (B) Vector-transduced GHOST R5 cells or GHOST R5 cells expressing FLAG-tagged FL IFITM2, Δ20 IFITM2, or IFITM3 were analyzed by Western blotting using the indicated antibodies. Note that the anti-IFITM2/3/Δ20 antibody recognizes FL IFITM2, Δ20 IFITM2, and IFITM3. (C) Experiments were similar to the experiments in Fig. 1E except that Δ20 IFITM2 expression in anti-CD3 and anti-CD28 antibody-activated CD4+ T cells from three different donors was analyzed. (D) Vector-transduced and Δ20 IFITM2-expressing Jurkat E6-1 R5 cells were labeled with anti-IFITM2/3/Δ20 primary and Alexa 488-conjugated secondary antibodies. Cells were then analyzed by flow cytometry. The histogram images of the Alexa 488 signal in the indicated cells and in cells labeled with a secondary antibody alone are shown. (E) Experiments were similar to the experiments in D except that anti-CD3 and anti-CD28 antibody-activated CD4+ T cells were analyzed. Experiments were similar to the experiments in C except that Δ20 IFITM2 expression in monocytes (F) and moDCs (G) was analyzed. Arrows indicate Δ20 IFITM2 bands. Note that the upper bands detected by the anti-IFITM2/3/Δ20 antibody could be FL IFITM2 or IFITM3. (H) Experiments were similar to the experiments in C except that anti-CD3 and anti-CD28 antibody-activated CD4+ T cells were treated with media, 1,000 U/mL IFN-β, or 1 μg/mL PHA for 2 d.

Article Snippet: Note that the upper bands detected by the anti-IFITM2/3/Δ20 antibody could be FL IFITM2 or IFITM3. ( H ) Experiments were similar to the experiments in C except that anti-CD3 and anti-CD28 antibody-activated CD4 + T cells were treated with media, 1,000 U/mL IFN-β, or 1 μg/mL PHA for 2 d. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Providers Immunogens Protein recognized Anti-IFITM1 (goat) R&D Systems (catalog no. AF4827) IFITM1 Met1-His36 IFITM1 Anti-IFITM2 (mouse) Proteintech (catalog no. 66137-1-Ig) IFITM2 FL FL IFITM2 Anti-IFITM2/3 (goat) R&D Systems (catalog no. AF4834) IFITM3 His3-His57 FL IFITM2, IFITM3, Δ20 IFITM2 (weakly) Anti-IFITM2/3/Δ20 (rabbit) Cell Signaling Technology (catalog no. 13530) IFITM2 around pro41 FL IFITM2, IFITM3, Δ20 IFITM2 Anti-IFITM1/2/3 (rabbit) ProSci (catalog no. 5807) IFITM1 FL IFITM1, FL IFITM2, IFITM3, Δ20 IFITM2 Open in a separate window Properties of anti-IFITM antibodies used in our studies Like other IFITM proteins, Δ20 IFITM2 expression was up-regulated by IFNs in CD4 + T cells, moDCs, and macrophages, whereas phytohemagglutinin (PHA), which has been widely used for propagating HIV-1, depleted its expression ( and and ).

Techniques: Expressing, Plasmid Preparation, Western Blot, Labeling, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Δ20 IFITM2 differentially restricts X4 and R5 HIV-1

doi: 10.1073/pnas.1619640114

Figure Lengend Snippet: Properties of anti-IFITM antibodies used in our studies

Article Snippet: Note that the upper bands detected by the anti-IFITM2/3/Δ20 antibody could be FL IFITM2 or IFITM3. ( H ) Experiments were similar to the experiments in C except that anti-CD3 and anti-CD28 antibody-activated CD4 + T cells were treated with media, 1,000 U/mL IFN-β, or 1 μg/mL PHA for 2 d. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Providers Immunogens Protein recognized Anti-IFITM1 (goat) R&D Systems (catalog no. AF4827) IFITM1 Met1-His36 IFITM1 Anti-IFITM2 (mouse) Proteintech (catalog no. 66137-1-Ig) IFITM2 FL FL IFITM2 Anti-IFITM2/3 (goat) R&D Systems (catalog no. AF4834) IFITM3 His3-His57 FL IFITM2, IFITM3, Δ20 IFITM2 (weakly) Anti-IFITM2/3/Δ20 (rabbit) Cell Signaling Technology (catalog no. 13530) IFITM2 around pro41 FL IFITM2, IFITM3, Δ20 IFITM2 Anti-IFITM1/2/3 (rabbit) ProSci (catalog no. 5807) IFITM1 FL IFITM1, FL IFITM2, IFITM3, Δ20 IFITM2 Open in a separate window Properties of anti-IFITM antibodies used in our studies Like other IFITM proteins, Δ20 IFITM2 expression was up-regulated by IFNs in CD4 + T cells, moDCs, and macrophages, whereas phytohemagglutinin (PHA), which has been widely used for propagating HIV-1, depleted its expression ( and and ).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Δ20 IFITM2 differentially restricts X4 and R5 HIV-1

doi: 10.1073/pnas.1619640114

Figure Lengend Snippet: Characterization of the subcellular distribution of Δ20 IFITM2. (A) A549 cells transduced to express the indicated IFITM proteins were labeled with anti-LAMP2, anti-IFITM1, or anti-IFITM2/3 antibodies. Labeled cells were imaged by confocal microscopy. (B) Percentages of voxels of IFITM proteins localizing to the plasma membrane are shown. Errors bars denote 1 SD (n = 20). (C) Experiments were similar to the experiments in Fig. 1 F and G except that vector-transduced, IFITM1-expressing, or IFITM3-expressing Jurkat E6-1 R5 cells were labeled with anti-IFITM1 or anti-IFITM2/3 antibodies. (D) Percentages of voxels of IFITM proteins localizing to the plasma membrane are shown. Errors bars denote 1 SD (n = 20). (E) Experiments were similar to the experiments in C except that unactivated and anti-CD3 and anti-CD28 antibody-activated CD4+ T cells were labeled with an anti-IFITM2/3/Δ20 antibody.

Article Snippet: Note that the upper bands detected by the anti-IFITM2/3/Δ20 antibody could be FL IFITM2 or IFITM3. ( H ) Experiments were similar to the experiments in C except that anti-CD3 and anti-CD28 antibody-activated CD4 + T cells were treated with media, 1,000 U/mL IFN-β, or 1 μg/mL PHA for 2 d. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antibody Providers Immunogens Protein recognized Anti-IFITM1 (goat) R&D Systems (catalog no. AF4827) IFITM1 Met1-His36 IFITM1 Anti-IFITM2 (mouse) Proteintech (catalog no. 66137-1-Ig) IFITM2 FL FL IFITM2 Anti-IFITM2/3 (goat) R&D Systems (catalog no. AF4834) IFITM3 His3-His57 FL IFITM2, IFITM3, Δ20 IFITM2 (weakly) Anti-IFITM2/3/Δ20 (rabbit) Cell Signaling Technology (catalog no. 13530) IFITM2 around pro41 FL IFITM2, IFITM3, Δ20 IFITM2 Anti-IFITM1/2/3 (rabbit) ProSci (catalog no. 5807) IFITM1 FL IFITM1, FL IFITM2, IFITM3, Δ20 IFITM2 Open in a separate window Properties of anti-IFITM antibodies used in our studies Like other IFITM proteins, Δ20 IFITM2 expression was up-regulated by IFNs in CD4 + T cells, moDCs, and macrophages, whereas phytohemagglutinin (PHA), which has been widely used for propagating HIV-1, depleted its expression ( and and ).

Techniques: Labeling, Confocal Microscopy, Clinical Proteomics, Membrane, Plasmid Preparation, 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: 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: Frontiers in molecular neuroscience

Article Title: Mutually Dependent Clustering of SynDIG4/PRRT1 and AMPA Receptor Subunits GluA1 and GluA2 in Heterologous Cells and Primary Neurons.

doi: 10.3389/fnmol.2022.788620

Figure Lengend Snippet: FIGURE 2 | The proline rich N-terminus of SD4 is dispensable for clustering with GluA1 and GluA2. (A) Schematic depicting the chimeric protein structures. Chimeras were generated expressing either (i) the N-terminus of SD4 and membrane domain of IFITM3 (SD4-NTD/IF-M) or (ii) the N-terminus of IFITM3 and membrane domain of SD4 (IF-NTD/SD4-M) (as shown in Table 1 of Methods). (B) Immunoblot of COS cell lysates transfected with SD4 and IFITM3 chimeras. β-tubulin was used as the loading control. (C) Representative confocal images depict either GluA1 expressed alone or co-expressed with either IF/SD4 chimera. Scale bar = 20 µm. (D,E) Graph depicts the mean cluster size of GluA1 puncta from the stratification of GluA1 co-localized or not co-localized with SD4-NTD/IF-M chimeras (D) or IF-NTD/SD4-M chimeras (E) compared with GluA1 alone. GluA1 alone (n = 10), GluA1 + SD4-NTD/IF-M (n = 12), and GluA1 + IF-NTD/SD4-M (n = 12). (F) Representative confocal images of GluA2 alone or co-expressed with IF/SD4 chimeras. Scale bar = 20 µm. (G,H) Graph depicts the mean cluster size of GluA2 puncta from the stratification of GluA2 co-localized or not co-localized with SD4-NTD/IF-M chimeras (G) or IF-NTD/SD4-M chimeras (H) compared with GluA2 alone. GluA2 alone (n = 13), GluA2 + SD4-NTD/IF-M (n = 12), and GluA2 + IF-NTD/SD4-M (n = 16). Data are represented as mean cluster size ± SEM; n.s. not significant; *p < 0.05; **p < 0.01; ****p < 0.0001; one-way ANOVA with post-hoc Tukey’s test.

Article Snippet: The following antibodies were used: mouse IgG1 antiGluA1 [Neuromab; Cat# 75-327; RRID: AB_2315840; Immunocytochemistry (ICC) 1:200; Immunoblotting (IB) 1:2,000]; mouse IgG2a anti-SynDIG4 (NeuroMab; Cat# 73-409; RRID: AB_2491106; ICC 1:200; IB 1:2,000); mouse IgG2a anti-SynDIG1 (NeuroMab; Cat# 75-251; RRID: AB_10999753; ICC 1:200); rabbit anti- Interferon-induced transmembrane protein 3 (IFITM3) (ProteinTech; ICC 1:200; IB 1:2,000); rat anti-hemagglutinin (HA) (Roche; ICC 1:50; IB 1:1,000); guinea pig anti-vGlut1 (EMD Millipore; ICC 1:500); Alexa 488- conjugated anti-mouse IgG2a (Molecular Probes; ICC 1:200); Alexa 594-conjugated anti-rat (Jackson ImmunoResearch; ICC 1:200); Alexa 555-cross adsorbed anti-mouse IgG1 (Invitrogen; ICC 1:500); Alexa 649-conjugated anti-guinea pig (Jackson ImmunoResearch: ICC 1:500); mouse anti-beta tubulin (MilliporeSigma; Clone: AA2; IB 1:5,000); goat horseradish peroxidase (HRP) conjugated anti-rat (Invitrogen; IB 1:5,000); and goat HRP-conjugated anti-mouse (Invitrogen; IB 1:10,000).

Techniques: Generated, Expressing, Membrane, Western Blot, Transfection, Control