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: Arthritis Research & Therapy

Article Title: A 2B adenosine receptor activity is reduced in neutrophils from patients with systemic sclerosis

doi: 10.1186/ar1468

Figure Lengend Snippet: Immunoblotting analysis of A 2A and A 2B adenosine receptors (ARs) from systemic sclerosis (SSc) neutrophils and controls. Cells obtained from 26 healthy volunteers and 26 SSc patients were lysed as described in the Methods section. Equal amounts of protein (50 μg) were separated on polyacrylamide gel, blotted and probed with 0.1 μg/ml rabbit anti-human A 2A AR or A 2B AR antibodies. Immunoreactive bands were visualized according to electrogenerated chemiluminescence protocol. A 2A and A 2B AR antibodies recognized immunoreactive bands of 45 kDa and 50 kDa, respectively. (a) Representative experiment performed on neutrophils from one healthy volunteer and one SSc patient. (b) Densitometric analysis of A 2A and A 2B AR immunoreactive bands from 26 healthy volunteers and 26 SSc patients. Graph bars: mean ± standard error of band density, normalized to β-actin. White bars are controls; grey bars are SSc patients.

Article Snippet: A 2A AR and A 2B AR antibodies were supplied by Alpha Diagnostic (San Antonio, TX, USA).

Techniques: Western Blot, Electrochemiluminescence