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: 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: 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: Native IFITM proteins were recognized by rabbit anti-IFITM2/3/Δ20 [which recognizes Δ20 IFITM2, FL-IFITM2, and IFITM3) (catalog no. 13530, 1:500; Cell Signaling Technology)] , mouse anti-IFITM2 (FL) [which only recognizes FL-IFITM2 (catalog no. 66137-1-Ig, 1 μg/mL; Proteintech)], goat anti-IFITM2/3 [which strongly binds to both IFITM2 and IFITM3 but weakly interacts with Δ20 IFITM2 (catalog no. AF4834, 1 μg/mL; R&D Systems)], goat anti-IFITM1 [which only interacts with IFITM1 (catalog no. AF4827, 1 μg/mL; R&D Systems)], and rabbit anti-IFITM1/2/3 [which recognizes IFITM1, IFITM2, IFITM3, and Δ20 IFITM2 (catalog no. 5807, 4 μg/mL; ProSci)] antibodies ( 10 ). lists the properties of anti-IFITM antibodies used in our studies.

Techniques: Quantitative RT-PCR, Nucleic Acid Electrophoresis, RNA Expression, RNA Sequencing Assay, 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 IFITM transcripts in human primary cells. (A) Schematic representation of three IFITM2 transcripts and primers used in our qRT-PCR analysis. Experiments were similar to the experiments in Fig. 1C except that expression of IFITM mRNA transcripts in anti-CD3 and anti-CD28 antibody-activated CD4+ T cells (B), unactivated CD4+ T cells (C), moDCs (D), M-CSF macrophages (E), or GM-CSF macrophages (F) from different donors was analyzed. In D–F, relative expression of the indicated IFITM transcripts to expression of Δ20 IFITM2 is shown. Expression of IFITM transcripts in CD4+ T cells and moDCs from donor 1 is shown in Fig. 1 C and D. Error bars denote 1 SD of triplicates or SEM of duplicates.

Article Snippet: Native IFITM proteins were recognized by rabbit anti-IFITM2/3/Δ20 [which recognizes Δ20 IFITM2, FL-IFITM2, and IFITM3) (catalog no. 13530, 1:500; Cell Signaling Technology)] , mouse anti-IFITM2 (FL) [which only recognizes FL-IFITM2 (catalog no. 66137-1-Ig, 1 μg/mL; Proteintech)], goat anti-IFITM2/3 [which strongly binds to both IFITM2 and IFITM3 but weakly interacts with Δ20 IFITM2 (catalog no. AF4834, 1 μg/mL; R&D Systems)], goat anti-IFITM1 [which only interacts with IFITM1 (catalog no. AF4827, 1 μg/mL; R&D Systems)], and rabbit anti-IFITM1/2/3 [which recognizes IFITM1, IFITM2, IFITM3, and Δ20 IFITM2 (catalog no. 5807, 4 μg/mL; ProSci)] antibodies ( 10 ). lists the properties of anti-IFITM antibodies used in our studies.

Techniques: Expressing, Quantitative RT-PCR

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: Native IFITM proteins were recognized by rabbit anti-IFITM2/3/Δ20 [which recognizes Δ20 IFITM2, FL-IFITM2, and IFITM3) (catalog no. 13530, 1:500; Cell Signaling Technology)] , mouse anti-IFITM2 (FL) [which only recognizes FL-IFITM2 (catalog no. 66137-1-Ig, 1 μg/mL; Proteintech)], goat anti-IFITM2/3 [which strongly binds to both IFITM2 and IFITM3 but weakly interacts with Δ20 IFITM2 (catalog no. AF4834, 1 μg/mL; R&D Systems)], goat anti-IFITM1 [which only interacts with IFITM1 (catalog no. AF4827, 1 μg/mL; R&D Systems)], and rabbit anti-IFITM1/2/3 [which recognizes IFITM1, IFITM2, IFITM3, and Δ20 IFITM2 (catalog no. 5807, 4 μg/mL; ProSci)] antibodies ( 10 ). lists the properties of anti-IFITM antibodies used in our studies.

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: Native IFITM proteins were recognized by rabbit anti-IFITM2/3/Δ20 [which recognizes Δ20 IFITM2, FL-IFITM2, and IFITM3) (catalog no. 13530, 1:500; Cell Signaling Technology)] , mouse anti-IFITM2 (FL) [which only recognizes FL-IFITM2 (catalog no. 66137-1-Ig, 1 μg/mL; Proteintech)], goat anti-IFITM2/3 [which strongly binds to both IFITM2 and IFITM3 but weakly interacts with Δ20 IFITM2 (catalog no. AF4834, 1 μg/mL; R&D Systems)], goat anti-IFITM1 [which only interacts with IFITM1 (catalog no. AF4827, 1 μg/mL; R&D Systems)], and rabbit anti-IFITM1/2/3 [which recognizes IFITM1, IFITM2, IFITM3, and Δ20 IFITM2 (catalog no. 5807, 4 μg/mL; ProSci)] antibodies ( 10 ). lists the properties of anti-IFITM antibodies used in our studies.

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: IFN enhances expression of Δ20 IFITM2. The moDCs (A), M-CSF macrophages (B), and GM-CSF macrophages (C) from different donors were treated with or without 1,000 U/mL IFN-β. Two days later, expression of IFITM mRNA transcripts was analyzed by qRT-PCR. Relative expression of the indicated IFITM mRNA in IFN-β–treated cells to expression in untreated controls is shown. Error bars denote 1 SD (n = 3). *P < 0.05 compared with controls.

Article Snippet: Native IFITM proteins were recognized by rabbit anti-IFITM2/3/Δ20 [which recognizes Δ20 IFITM2, FL-IFITM2, and IFITM3) (catalog no. 13530, 1:500; Cell Signaling Technology)] , mouse anti-IFITM2 (FL) [which only recognizes FL-IFITM2 (catalog no. 66137-1-Ig, 1 μg/mL; Proteintech)], goat anti-IFITM2/3 [which strongly binds to both IFITM2 and IFITM3 but weakly interacts with Δ20 IFITM2 (catalog no. AF4834, 1 μg/mL; R&D Systems)], goat anti-IFITM1 [which only interacts with IFITM1 (catalog no. AF4827, 1 μg/mL; R&D Systems)], and rabbit anti-IFITM1/2/3 [which recognizes IFITM1, IFITM2, IFITM3, and Δ20 IFITM2 (catalog no. 5807, 4 μg/mL; ProSci)] antibodies ( 10 ). lists the properties of anti-IFITM antibodies used in our studies.

Techniques: Expressing, Quantitative RT-PCR

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: Native IFITM proteins were recognized by rabbit anti-IFITM2/3/Δ20 [which recognizes Δ20 IFITM2, FL-IFITM2, and IFITM3) (catalog no. 13530, 1:500; Cell Signaling Technology)] , mouse anti-IFITM2 (FL) [which only recognizes FL-IFITM2 (catalog no. 66137-1-Ig, 1 μg/mL; Proteintech)], goat anti-IFITM2/3 [which strongly binds to both IFITM2 and IFITM3 but weakly interacts with Δ20 IFITM2 (catalog no. AF4834, 1 μg/mL; R&D Systems)], goat anti-IFITM1 [which only interacts with IFITM1 (catalog no. AF4827, 1 μg/mL; R&D Systems)], and rabbit anti-IFITM1/2/3 [which recognizes IFITM1, IFITM2, IFITM3, and Δ20 IFITM2 (catalog no. 5807, 4 μg/mL; ProSci)] antibodies ( 10 ). lists the properties of anti-IFITM antibodies used in our studies.

Techniques: Labeling, Confocal Microscopy, Membrane, Plasmid Preparation, Expressing

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: Δ20 IFITM2 differentially restricts replication of X4 and R5 HIV-1. Jurkat E6-1 R5 cells expressing the indicated IFITM proteins were incubated with 100 ng of p24 antigen X4-tropic NL4-3 (A) or R5-tropic AD8 (B) virus. Supernatants were harvested at the indicated time points, and virus titers were measured by p24 ELISA. Numbers indicate p24 values (mean ± SD × 105; n = 3) detected in the supernatants of vector-transduced and Δ20 IFITM2-expressing cells. Experiments similar to the experiments in A and B, except that the indicated X4 (C) or R5 (D) viruses were used, were performed. Numbers indicate p24 values (mean ± SD; n = 3). Vector-transduced GHOST R5 cells or GHOST R5 cells expressing Δ20 IFITM2 were incubated with the indicated replicating X4 (E) or R5 (F) virus. Two days later, infected cells were harvested and analyzed by flow cytometry. The relative infectivity was determined as the percentage of GFP+ cells normalized to the percentage of vector-transduced cells. Error bars denote 1 SEM of duplicates.

Article Snippet: Native IFITM proteins were recognized by rabbit anti-IFITM2/3/Δ20 [which recognizes Δ20 IFITM2, FL-IFITM2, and IFITM3) (catalog no. 13530, 1:500; Cell Signaling Technology)] , mouse anti-IFITM2 (FL) [which only recognizes FL-IFITM2 (catalog no. 66137-1-Ig, 1 μg/mL; Proteintech)], goat anti-IFITM2/3 [which strongly binds to both IFITM2 and IFITM3 but weakly interacts with Δ20 IFITM2 (catalog no. AF4834, 1 μg/mL; R&D Systems)], goat anti-IFITM1 [which only interacts with IFITM1 (catalog no. AF4827, 1 μg/mL; R&D Systems)], and rabbit anti-IFITM1/2/3 [which recognizes IFITM1, IFITM2, IFITM3, and Δ20 IFITM2 (catalog no. 5807, 4 μg/mL; ProSci)] antibodies ( 10 ). lists the properties of anti-IFITM antibodies used in our studies.

Techniques: Expressing, Incubation, Virus, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Infection, 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: Δ20 IFITM2 differentially restricts X4 and R5 HIV-1 in Jurkat E6-1 R5 cells. (A) Expression of IFITM proteins in Jurkat E6-1 R5 cells used in Fig. 2 A–D was analyzed by Western blotting using the indicated antibodies. (B) Vector-transduced, rs1059091-A Δ20 IFITM2-expressing, or rs1059091-G Δ20 IFITM2-expressing Jurkat E6-1 R5 cells were infected with NL4-3-IeG (X4) or JRFL-IeG (R5) HIV-1. Three days later, cells were harvested and analyzed by flow cytometry. The relative infectivity was determined as the percentage of GFP+ cells normalized to the percentage of vector-transduced control cells. (C) Same aliquots of cells used in B were analyzed by Western blotting using the indicated antibodies. (D) Vector-transduced Jurkat E6-1 R5 cells or Jurkat E6-1 R5 cells stably expressing the indicated IFITM proteins were labeled with anti-CD4, anti-CXCR4, or anti-CCR5 antibodies and then analyzed by flow cytometry. Histogram images are shown. (E) Same aliquots of cells used in Fig. 2 E and F were analyzed by Western blotting using the indicated antibodies. (F) Same aliquots of cells used in Fig. 3 A and B were analyzed by Western blotting. (G) Same aliquots of cells used in Fig. 3D were analyzed by Western blotting using the indicated antibodies.

Article Snippet: Native IFITM proteins were recognized by rabbit anti-IFITM2/3/Δ20 [which recognizes Δ20 IFITM2, FL-IFITM2, and IFITM3) (catalog no. 13530, 1:500; Cell Signaling Technology)] , mouse anti-IFITM2 (FL) [which only recognizes FL-IFITM2 (catalog no. 66137-1-Ig, 1 μg/mL; Proteintech)], goat anti-IFITM2/3 [which strongly binds to both IFITM2 and IFITM3 but weakly interacts with Δ20 IFITM2 (catalog no. AF4834, 1 μg/mL; R&D Systems)], goat anti-IFITM1 [which only interacts with IFITM1 (catalog no. AF4827, 1 μg/mL; R&D Systems)], and rabbit anti-IFITM1/2/3 [which recognizes IFITM1, IFITM2, IFITM3, and Δ20 IFITM2 (catalog no. 5807, 4 μg/mL; ProSci)] antibodies ( 10 ). lists the properties of anti-IFITM antibodies used in our studies.

Techniques: Expressing, Western Blot, Plasmid Preparation, Infection, Flow Cytometry, Control, Stable Transfection, Labeling

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: Δ20 IFITM2 differentially restricts entry of X4 and R5 HIV-1. (A and B) Vector-transduced GHOST R5 cells or GHOST R5 cells expressing Δ20 IFITM2 were incubated with NL4-3-ΔE pseudotyped with the indicated viral entry glycoproteins. Two days later, cells were harvested and analyzed by flow cytometry. The relative infectivity was determined as the percentage of GFP+ cells normalized to the percentage of vector-transduced control cells. LCMV, lymphocytic choriomeningitis virus. (C) Vector-transduced Jurkat E6-1 R5 cells or Jurkat E6-1 R5 cells stably expressing Δ20 IFITM2 were incubated with pLenti-based HIV-1–GFP pseudotyped with env proteins from the indicated HIV-1 strains. Two days later, cells were harvested and analyzed by flow cytometry. The relative infectivity was determined as the percentage of GFP+ cells normalized to the percentage of vector-transduced cells. MLV, murine leukemia virus. (D) Experiments similar to the experiments in C, except that cells expressing different amounts of Δ20 IFITM2, were used. Numbers indicate percentages of infected control cells. (E) Primary moDCs were transfected with scrambled siRNA or siRNA targeting IFITM2 transcripts. Two days later, cells were incubated with NL4-3-IeG (X4) or JRFL-IeG (R5). One day later, supernatants were harvested and virus titers were determined by p24 ELISA. Numbers indicate the p24 values detected in the supernatants of scrambled siRNA-transfected cells. Experiments were performed at least three times with similar results. (F) Same aliquots of cells used in E were analyzed for mRNA expression of the indicated IFITM transcripts using qRT-PCR. Expression of the indicated IFITM mRNA in IFITM2 siRNA-transfected cells relative to expression of the indicated IFITM mRNA in scrambled siRNA transfected controls is shown. Error bars denote 1 SD (n = 3). *P < 0.05 compared with controls.

Article Snippet: Native IFITM proteins were recognized by rabbit anti-IFITM2/3/Δ20 [which recognizes Δ20 IFITM2, FL-IFITM2, and IFITM3) (catalog no. 13530, 1:500; Cell Signaling Technology)] , mouse anti-IFITM2 (FL) [which only recognizes FL-IFITM2 (catalog no. 66137-1-Ig, 1 μg/mL; Proteintech)], goat anti-IFITM2/3 [which strongly binds to both IFITM2 and IFITM3 but weakly interacts with Δ20 IFITM2 (catalog no. AF4834, 1 μg/mL; R&D Systems)], goat anti-IFITM1 [which only interacts with IFITM1 (catalog no. AF4827, 1 μg/mL; R&D Systems)], and rabbit anti-IFITM1/2/3 [which recognizes IFITM1, IFITM2, IFITM3, and Δ20 IFITM2 (catalog no. 5807, 4 μg/mL; ProSci)] antibodies ( 10 ). lists the properties of anti-IFITM antibodies used in our studies.

Techniques: Plasmid Preparation, Expressing, Incubation, Flow Cytometry, Infection, Control, Virus, Stable Transfection, Transfection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

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: Infection of X4, but not R5, HIV-1 was enhanced upon depletion of IFITM2. Primary moDCs (A) or M-CSF–derived macrophages (C) were transfected with scrambled siRNA or siRNA targeting IFITM2 (both FL- and Δ20 IFITM2) transcripts. Two days later, cells were incubated with NL4-3-IeG (X4) or JRFL-IeG (R5) HIV-1. Cells were harvested 24 h after infection. HIV-1 early and late reverse transcripts were analyzed by real-time PCR. Reverse transcripts in IFITM2 siRNA-transfected cells relative to reverse transcripts in scrambled siRNA-transfected controls are shown. (B and D) Expression of IFITM2 (both FL- and Δ20 IFITM2) mRNA in cells used in A and C was assayed by qRT-PCR. Experiments were performed at least twice with similar results. Error bars denote 1 SEM of duplicates. (E) Same aliquots of cells used in Fig. 3 E and F were analyzed by Western blotting using the indicated antibodies.

Article Snippet: Native IFITM proteins were recognized by rabbit anti-IFITM2/3/Δ20 [which recognizes Δ20 IFITM2, FL-IFITM2, and IFITM3) (catalog no. 13530, 1:500; Cell Signaling Technology)] , mouse anti-IFITM2 (FL) [which only recognizes FL-IFITM2 (catalog no. 66137-1-Ig, 1 μg/mL; Proteintech)], goat anti-IFITM2/3 [which strongly binds to both IFITM2 and IFITM3 but weakly interacts with Δ20 IFITM2 (catalog no. AF4834, 1 μg/mL; R&D Systems)], goat anti-IFITM1 [which only interacts with IFITM1 (catalog no. AF4827, 1 μg/mL; R&D Systems)], and rabbit anti-IFITM1/2/3 [which recognizes IFITM1, IFITM2, IFITM3, and Δ20 IFITM2 (catalog no. 5807, 4 μg/mL; ProSci)] antibodies ( 10 ). lists the properties of anti-IFITM antibodies used in our studies.

Techniques: Infection, Derivative Assay, Transfection, Incubation, Real-time Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Western Blot

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: Δ20 IFITM2 colocalizes with CXCR4 and CCR5. (A) GHOST R5 cells expressing Δ20 IFITM2 were fixed and labeled with the indicated antibodies and DAPI. Cells were imaged by confocal microscopy. (Insets) Enlarged images are shown. (B) Expression of Δ20 IFITM2 and CCR5 variants in cells used in Fig. 4 D–F was analyzed by Western blotting. (C) Expression of Δ20 IFITM2 and CXCR4 variants in cells used in Fig. 4 B and C was analyzed by Western blotting.

Article Snippet: Native IFITM proteins were recognized by rabbit anti-IFITM2/3/Δ20 [which recognizes Δ20 IFITM2, FL-IFITM2, and IFITM3) (catalog no. 13530, 1:500; Cell Signaling Technology)] , mouse anti-IFITM2 (FL) [which only recognizes FL-IFITM2 (catalog no. 66137-1-Ig, 1 μg/mL; Proteintech)], goat anti-IFITM2/3 [which strongly binds to both IFITM2 and IFITM3 but weakly interacts with Δ20 IFITM2 (catalog no. AF4834, 1 μg/mL; R&D Systems)], goat anti-IFITM1 [which only interacts with IFITM1 (catalog no. AF4827, 1 μg/mL; R&D Systems)], and rabbit anti-IFITM1/2/3 [which recognizes IFITM1, IFITM2, IFITM3, and Δ20 IFITM2 (catalog no. 5807, 4 μg/mL; ProSci)] antibodies ( 10 ). lists the properties of anti-IFITM antibodies used in our studies.

Techniques: Expressing, Labeling, Confocal Microscopy, Western Blot

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: C-terminal region of CCR5 contributes to the resistance of HIV-1 to Δ20 IFITM2-mediated restriction. (A) Schematic representation of the CCR5 variant, CCR5DM, and CCR5/CXCR4 chimeras, CCR5CXCR4 and CXCR4CCR5, used in our experiments. Experiments were similar to the experiments in Fig. 3 A and B except that GHOST R5 cells expressing WT CXCR4 (B) or CXCR4CCR5 (C) were used. Experiments were similar to the experiments in Fig. 3 A and B except that GHOST X4 cells expressing WT CCR5 (D), CCR5DM (E), or CCR5CXCR4 (F) were used. Experiments were performed at least three times with similar results. Error bars denote 1 SD (n = 3). *P < 0.05 compared with controls.

Article Snippet: Native IFITM proteins were recognized by rabbit anti-IFITM2/3/Δ20 [which recognizes Δ20 IFITM2, FL-IFITM2, and IFITM3) (catalog no. 13530, 1:500; Cell Signaling Technology)] , mouse anti-IFITM2 (FL) [which only recognizes FL-IFITM2 (catalog no. 66137-1-Ig, 1 μg/mL; Proteintech)], goat anti-IFITM2/3 [which strongly binds to both IFITM2 and IFITM3 but weakly interacts with Δ20 IFITM2 (catalog no. AF4834, 1 μg/mL; R&D Systems)], goat anti-IFITM1 [which only interacts with IFITM1 (catalog no. AF4827, 1 μg/mL; R&D Systems)], and rabbit anti-IFITM1/2/3 [which recognizes IFITM1, IFITM2, IFITM3, and Δ20 IFITM2 (catalog no. 5807, 4 μg/mL; ProSci)] antibodies ( 10 ). lists the properties of anti-IFITM antibodies used in our studies.

Techniques: Variant Assay, Expressing