Journal: DNA repair

Article Title: Persistent 3′-phosphate termini and increased cytotoxicity of radiomimetic DNA double-strand breaks in cells lacking polynucleotide kinase/phosphatase despite presence of an alternative 3′-phosphatase

doi: 10.1016/j.dnarep.2018.05.002

Figure Lengend Snippet: Dependence of 3′-phosphatase and 5′-kinase activities on XRCC4. A. Plasmid-length 5′-hydroxyl DSB substrate for 5′-phosphorylation. Bold lettering indicates oligomeric duplex that was ligated to pUC19. SmaI releases a labeled 16-base oligonucleotide. B. Upper gel: The 3′ overhang 3′-phosphate substrate (Fig. 4A) was incubated in HCT116 WT, XRCC4−/− or PNKP−/− extracts for 1 hr, cut with TaqαI and BstXI and 3′-dephosphorylation was assessed as in Fig. 3. Lower gel: The 5′-hydroxyl substrate shown in (A.) was incubated in the same extracts for 4 hr, cut with SmaI, and analyzed by gel electrophoresis to assess 5′-phosphorylation. C. The same substrate was incubated for 6 hr in whole-cell extracts of HCT116 and HeLa cells or their PNKP-deficient derivatives, and 5′-phosphorylation was similarly determined. Error bars show mean ± SEM for 3 experiments.

Article Snippet: The oligonucleotides were purchased from IDT and in some cases 5′- 32 P labeled using a 3′-phosphatase-free polynucleotide kinase (NEB).

Techniques: Plasmid Preparation, Labeling, Incubation, De-Phosphorylation Assay, Nucleic Acid Electrophoresis

Journal: DNA repair

Article Title: Persistent 3′-phosphate termini and increased cytotoxicity of radiomimetic DNA double-strand breaks in cells lacking polynucleotide kinase/phosphatase despite presence of an alternative 3′-phosphatase

doi: 10.1016/j.dnarep.2018.05.002

Figure Lengend Snippet: Effect of DNA-PK inhibitor NU7441 (DNA-PKi) on dephosphorylation in whole-cell extracts and detection of 3′-phosphatase in nuclear extracts. A. Gel showing 3′-dephosphorylation of the 3′-recessed DSB substrate in HCT116 WT and PNKP−/− extracts. No-inhibitor control reactions were performed both without (“C”) and with (“D”) DMSO, which is the solvent for DNA-PKi. B. and C. Quantitation of 3′-dephosphorylation for recessed and overhang substrates in HCT116 WT and PNKP−/− extracts. D. Gel showing 3′-dephosphorylation of the 3′ overhang substrate in HeLa WT and PNKP−/−/− extracts. E. and F. Quantitation of 3′-dephosphorylation for recessed and overhang substrates in HeLa WT and PNKP−/−/− extracts. G. Gel showing 3′-dephosphorylation of the 3′ overhang substrate in nuclear extracts of HCT116 WT and PNKP−/− cells. H. Quantitation of 3′-dephosphorylation in nuclear extracts of HCT116 WT and PNKP−/− cells. I. Quantitation of 3′-dephosphorylation in nuclear extracts of HeLa WT and PNKP−/−/− cells. Shorter labeled products resulting from subsequent 3′→5′ exonucleolytic resection were included in calculation of dephosphorylation. Error bars = mean ± SEM for 3 experiments, except H. and I., 2 experiments.

Article Snippet: The oligonucleotides were purchased from IDT and in some cases 5′- 32 P labeled using a 3′-phosphatase-free polynucleotide kinase (NEB).

Techniques: De-Phosphorylation Assay, Quantitation Assay, Labeling

Journal: The Japanese Dental Science Review

Article Title: Salivary SARS-CoV-2 RNA for diagnosis of COVID-19 patients: a systematic revisew and meta-analysis of diagnostic accuracy ☆

doi: 10.1016/j.jdsr.2023.06.004

Figure Lengend Snippet: Characteristics of the population on the eligible studies included.

Article Snippet: Echavarria et al. , Argentina , 174 (hospital -emergency) , 31 , , , , , , , , , , , , , , , , , , , , Symptomatic , Viral RNA detection with PCR from NPS , Quick‐RNA™ Viral Kit, Zymo Research Corp , CFX 96 Deep Well™ Real Time System (BioRad) , The E gene , Nasopharyngeal , Whole saliva.

Techniques: RNA Extraction, RNA Detection, Real-time Polymerase Chain Reaction, Purification, Diagnostic Assay, Nucleic Acid Purification, Amplification, Quantitative RT-PCR, Multiplex Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Genetics

Article Title: A Novel α-Galactosidase A Splicing Mutation Predisposes to Fabry Disease

doi: 10.3389/fgene.2019.00060

Figure Lengend Snippet: Alternative splicing of GLA (c.801+1G > A). (A) mRNA from the patient blood was extracted and amplified by RT-PCR for alternative splicing of GLA (c.801+1G > A). RT-PCR of the region that includes exons 5 and 6 was performed and the PCR products were separated by gel electrophoresis. (B) Sanger sequencing analysis for the alternative splicing products. The sequence of the upper band reveals a 36-nucleotide (nt) insertion at the junction between exons 5 and 6 of the GLA cDNA compare to wild type. (C) Schematic representation of GLA . Uppercase letters indicate the exonic sequences, whereas lowercase letters indicate the intronic sequences. The encoded amino acids are depicted in single-letter code. The changed nucleotide in mutant GLA cDNA is labeled by underscore. (D) qRT-PCR analysis performed on total RNA obtained from blood samples of patient III4 and three healthy volunteers individuals. Levels were normalized to the amount of GAPDH. Data represent the mean ± SE of three independent measurements performed in triplicate ( ∗∗ p < 0.01).

Article Snippet: The following antibodies were used: GFP mouse monoclonal antibody (Proteintech, Cat. No. 66002-1-Ig), GAPDH mouse monoclonal antibody (Proteintech, Cat. No. 60004-1-Ig).

Techniques: Alternative Splicing, Amplification, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Sequencing, Mutagenesis, Labeling, Quantitative RT-PCR

Journal: Frontiers in Genetics

Article Title: A Novel α-Galactosidase A Splicing Mutation Predisposes to Fabry Disease

doi: 10.3389/fgene.2019.00060

Figure Lengend Snippet: Enzyme activity of the mutant GLA. (A) Images taking from bright and fluorescence fields were used to show the similar transfection efficiency of GFP, GFP-GLA-WT, or GFP-GLA-MT plasmids in HEK293T cells. (B) Western blot analysis for HEK293T lysates transfected with GFP, GFP-GLA-WT, or GFP-GLA-MT plasmids. GAPDH was used as loading control. (C) The result of enzyme activity assay from HEK293T cells transfected with wild type or mutant GLA plasmids. Data were presented as the mean ± SD from three independent experiments; ∗∗∗ p < 0.001.

Article Snippet: The following antibodies were used: GFP mouse monoclonal antibody (Proteintech, Cat. No. 66002-1-Ig), GAPDH mouse monoclonal antibody (Proteintech, Cat. No. 60004-1-Ig).

Techniques: Activity Assay, Mutagenesis, Fluorescence, Transfection, Western Blot, Control, Enzyme Activity Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Inhibition of miR-29b-1-5p ameliorates lipopolysaccharide (LPS)-induced septic myocardial injury in mice (A) Mice received a single intraperitoneal injection of 25 mg/kg LPS (lethal dose) or a tail vein injection of 80 mg/kg miR-29b-1-5p/NC antagomir for 3 consecutive days (24 h later receiving LPS injection), after which the survival conditions were assessed for 96 h, and survival rate curves were generated. An equal amount of saline was injected into the control group. n=20. Mice received a single intraperitoneal injection of 10 mg/kg LPS or a tail vein injection of 80 mg/kg miR-29b-1-5p/NC antagomir for 3 consecutive days (24 h later receiving LPS injection). (B) Twenty-four hours after LPS injection, echocardiography tests were performed. (C,D) Mice were euthanized, and histological analysis of myocardial tissue injury was performed via hematoxylin and eosin (HE) staining and Masson’s trichrome staining. Scale bar: 50 μm. (F,G) The cardiac injury score and fibrotic area were assessed via HE and Masson’s trichrome staining, respectively. (E,H) α-Smooth muscle actin (α-SMA) expression in mouse myocardial tissues was determined via immunohistochemistry (IHC) staining. Scale bar: 20 μm. (I‒L) Tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and cardiac troponin I (cTnI) levels in mouse serum were determined by enzyme-linked immunosorbent assay (ELISA). (M) The miR-29b-1-5p expression level in mouse myocardial tissue was determined by real-time quantitative reverse transcription PCR (qRT-PCR). n=6. (N) The protein levels of TLR2, TLR4, TFEB, LAMP1 and LC3II in mouse myocardial tissues were determined by western blot analysis. n=3. **P<0.01 vs the control group; #P<0.05 and ##P<0.01 vs. the LPS+NC antagomir group.

Article Snippet: After separation by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 50 μg of total protein was transferred to PVDF membranes (Millipore, Billerica, USA), which were blocked with 5% skim milk in TBST solution for 2 h. Then the membranes were incubated overnight at 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Inhibition, Injection, Generated, Saline, Staining, Expressing, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Inhibition of miR-29b-1-5p ameliorates CLP-induced septic myocardial injury in mice (A) Mice were subjected to CLP surgery or tail vein injection of 80 mg/kg miR-29b-1-5p/NC antagomir for 3 consecutive days (24 h later receiving CLP surgery), after which the survival conditions were assessed for 96 h, and survival rate curves were generated. n=20. A sham operation was performed on the control group. n=20. (B) Twenty-four hours after CLP surgery, echocardiography was performed. (C,D) Mice were euthanized, and histological analysis of myocardial tissue injury was performed via HE staining and Masson’s trichrome staining. Scale bar: 50 μm. (F,G) The cardiac injury score and fibrotic area were assessed via HE and Masson’s trichrome staining, respectively. (E,H) The protein levels and distribution of α-SMA in mouse myocardial tissues were examined using IHC staining. Scale bar: 20 μm. (I‒L) TNF-α, IL-1β, IL-6 and cTnI levels in mouse serum were determined by ELISA. (M) The miR-29b-1-5p expression level in mouse myocardial tissue was determined by qRT-PCR. n=6. (N) The protein levels of TLR2, TLR4, TFEB, LAMP1 and LC3II in mouse myocardial tissues were determined by western blot analysis. n=3. **P<0.01 vs the control group; #P<0.05 and ##P<0.01 vs the CLP+NC antagomir group.

Article Snippet: After separation by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 50 μg of total protein was transferred to PVDF membranes (Millipore, Billerica, USA), which were blocked with 5% skim milk in TBST solution for 2 h. Then the membranes were incubated overnight at 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Inhibition, Injection, Generated, Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Inhibition of miR-29b-1-5p ameliorates LPS-induced cardiomyocyte dysfunction in mice in vitro (A) Twenty-four hours after transfecting HL-1 cells with the NC or miR-29b-1-5p antagomir, LPS (1 μg/mL) stimulation was applied for another 24 h, and miR-29b-1-5p expression was validated in each group by qRT-PCR. (B) After LPS treatment for 0, 24, 48, or 72 h, MTT assay was performed to examine the viability of the HL-1 cells. (C) Flow cytometry was used to evaluate apoptosis in HL-1 cells. (D‒F) TNF-α, IL-1β, and IL-6 levels in HL-1 cell supernatants were measured by enzyme-linked immunosorbent assay (ELISA). (G‒I) Western blot analysis was used to measure Bax, Bcl-2, cleaved caspase-3, pro-caspase-3, TLR2, TLR4, TFEB, LAMP1 and LC3II protein levels in HL-1 cells. n=3. **P<0.01 vs the control group; ##P<0.01 vs the LPS+NC antagomir group.

Article Snippet: After separation by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 50 μg of total protein was transferred to PVDF membranes (Millipore, Billerica, USA), which were blocked with 5% skim milk in TBST solution for 2 h. Then the membranes were incubated overnight at 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Inhibition, In Vitro, Expressing, Quantitative RT-PCR, MTT Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Silencing of TERF2 partially reverses the ameliorative effect of miR-29b-1-5p inhibition on LPS-induced cardiomyocyte apoptosis and inflammation (A) HL-1 cells were transfected with sh-TERF2 or sh-NC vector. Forty-eight hours later, HL-1 cells were collected to detect the protein expression of TERF2. (B‒I) HL-1 cells were transfected with sh-TERF2 or the miR-29b-1-5p antagomir for 24 h, followed by stimulation with LPS. MTT assay was performed to examine HL-1 cell viability (B). Flow cytometry was performed to evaluate HL-1 cell apoptosis (C). ELISA was also conducted to evaluate TNF-α, IL-1β, and IL-6 levels in HL-1 cells (D‒F). Western blot analysis was conducted to determine the protein levels of TERF2, cleaved PARP, cleaved caspase-3, pro-caspase-3, TLR2, TLR4, TFEB, LAMP1 and LC3II in HL-1 cells (G–I). **P<0.01 vs LPS+NC antagomir+sh-NC; #P<0.05, ##P<0.01 vs LPS+miR-29b-1-5p antagomir+sh-TERF2.

Article Snippet: After separation by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 50 μg of total protein was transferred to PVDF membranes (Millipore, Billerica, USA), which were blocked with 5% skim milk in TBST solution for 2 h. Then the membranes were incubated overnight at 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Inhibition, Transfection, Plasmid Preparation, Expressing, MTT Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, 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: 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: Oncogene

Article Title: Diallyl trisulfide-induced apoptosis in human prostate cancer cells involves c-Jun N-terminal kinase and extracellular-signal regulated kinase-mediated phosphorylation of Bcl-2.

doi: 10.1038/sj.onc.1207747

Figure Lengend Snippet: Figure 4 Immunoblotting for (a) phosphorylated (activated), and (b) total ERK1/2 (top panel), p38 (middle panel) and JNK1 (bottom panel) using lysates from PC-3 cells treated with 40 mM DATS for the indicated times. Equal amounts of lysate proteins were used for gel electrophoresis. Blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. (c) ERK1/ 2 and JNK kinase activities using lysates from PC-3 cells treated with DMSO (control) and 40 mM DATS for 30 min (ERK1/2) or 4 h (JNK). The experiment was repeated two or three times to ensure reproducibility

Article Snippet: Antibodies against Bax (sc-493), BID (sc-6538), Thr56 phosphorylated Bcl-2 (sc-16321), ERK1 (sc-93), phospho-(Tyr204)-ERK1/2 (sc-7383), JNK1 (sc-571), phospho-(Thr183/Tyr185)-JNK (sc-12882), p38 (sc-535) and phospho-(Tyr182)-p38 (sc-7973) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), antibody against Bcl-2 was from DAKO (Carpinteria, CA, USA), antibody specific for cleaved caspase-3 (catalogue # 9661) was from Cell Signaling Technology (Beverly, MA, USA), antibody against Oncogene procaspase-9 (catalogue # 556585) was from BD Pharmingen, Palo Alto, CA, USA and the antibody against actin was from Oncogene Research Products (Boston, MA, USA).

Techniques: Western Blot, Nucleic Acid Electrophoresis, Control

Journal: Oncogene

Article Title: Diallyl trisulfide-induced apoptosis in human prostate cancer cells involves c-Jun N-terminal kinase and extracellular-signal regulated kinase-mediated phosphorylation of Bcl-2.

doi: 10.1038/sj.onc.1207747

Figure Lengend Snippet: Figure 7 (a) Effects of OSCs on proliferation of DU145 cells determined by trypan blue dye exclusion assay. DU145 cells were treated with DMSO (control) or 40 mM DAS, DADS or DATS for 24, 48 or 72 h. Data are mean7s.e. (n ¼ 3). *Significantly different compared with control (Po0.05) by one-way ANOVA. (b) Effects of OSCs on apoptosis induction in DU145 cells determined by DAPI assay. DU145 cells were treated with DMSO (control) or 40 mM DAS, DADS or DATS for 24 h. Data are mean7s.e. (n ¼ 3). *Significantly different compared with control (Po0.05) by one-way ANOVA. (c) Immunoblotting for phospho-JNK1/2 (top panel) and phospho-ERK1/2 (middle panel) using lysates from DU145 cells treated with 40 mM DATS for the indicated times. Blots were stripped and reprobed with anti-actin antibody (bottom panel) to ensure equal protein loading. Results were similar in three independent experiments, and representative data from a single experiment are shown. (d) Effect of DATS treatment (40 mM, 30 min) on kinase activity of ERK1/2 and JNKs determined by monitoring phosphorylation of Elk-1 (Ser383) and c-Jun (Ser63), respectively. The experiment was repeated two times, and the results were comparable. (e) Effect of MEK1 inhibitor U0126 on apoptosis induction by DATS in DU145 cells determined by quantitation of cytoplasmic histone-associated DNA fragments. DU145 cells were pretreated with U0126 (10 mM) for 2 h, and then treated with 40 mM DATS for 24 h in the presence of the inhibitor. Cells were collected, and processed for analysis of cytoplasmic histone-associated DNA fragments. Experiment was repeated three times, and the results were similar. Representative data (mean7s.e.) from a single experiment are shown. *Significantly different compared with DMSO-treated control, Po0.05. (f) Effect of JNK inhibitor SP600125 on apoptosis induction by DATS in DU145 cells. DU145 cells were pretreated with SP600125 (10 mM) for 2 h, and then treated with 40 mM DATS for 24 h in the presence of the inhibitor. Cells were collected, and processed for analysis of cytoplasmic histone-associated DNA fragments. Experiment was repeated three times, and the results were similar. Representative data (mean7s.e.) from a single experiment are shown. *Significantly different compared with DMSO-treated control, Po0.05, and þSignificantly different compared with DATS alone group, Po0.05. Statistical significance was assessed by one- way ANOVA followed by Bonferroni’s multiple comparison test

Article Snippet: Antibodies against Bax (sc-493), BID (sc-6538), Thr56 phosphorylated Bcl-2 (sc-16321), ERK1 (sc-93), phospho-(Tyr204)-ERK1/2 (sc-7383), JNK1 (sc-571), phospho-(Thr183/Tyr185)-JNK (sc-12882), p38 (sc-535) and phospho-(Tyr182)-p38 (sc-7973) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), antibody against Bcl-2 was from DAKO (Carpinteria, CA, USA), antibody specific for cleaved caspase-3 (catalogue # 9661) was from Cell Signaling Technology (Beverly, MA, USA), antibody against Oncogene procaspase-9 (catalogue # 556585) was from BD Pharmingen, Palo Alto, CA, USA and the antibody against actin was from Oncogene Research Products (Boston, MA, USA).

Techniques: Exclusion Assay, Control, Western Blot, Activity Assay, Phospho-proteomics, Quantitation Assay, Comparison

Journal: Oncogene

Article Title: Diallyl trisulfide-induced apoptosis in human prostate cancer cells involves c-Jun N-terminal kinase and extracellular-signal regulated kinase-mediated phosphorylation of Bcl-2.

doi: 10.1038/sj.onc.1207747

Figure Lengend Snippet: Figure 8 (a) Immunoblotting for catalase, phospho-JNK1/2, and phospho-ERK1/2 using lysates from Ad-EGFP or Ad-catalase DU145 cells treated with DMSO (control) or 40 mM DATS for 30 min. The blots were stripped and reprobed with anti-actin antibodies to ensure equal protein loading. Experiment was repeated at least three times, and the results were comparable. Data from a representative experiment are shown. (b) Analysis of cytoplasmic histone-associated DNA fragments following a 24 h treatment of Ad-EGFP or Ad-catalase DU145 cells with DMSO (control) or 40 mM DATS. Experiment was repeated three times with similar results, and representative data from a single experiment are shown. Data are mean7s.e. (n ¼ 3). *Cytoplasmic histone-associated DNA fragmentation was significantly higher in DATS-treated cells compared with DMSO control in Ad-EGFP but not in Ad-catalase

Article Snippet: Antibodies against Bax (sc-493), BID (sc-6538), Thr56 phosphorylated Bcl-2 (sc-16321), ERK1 (sc-93), phospho-(Tyr204)-ERK1/2 (sc-7383), JNK1 (sc-571), phospho-(Thr183/Tyr185)-JNK (sc-12882), p38 (sc-535) and phospho-(Tyr182)-p38 (sc-7973) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), antibody against Bcl-2 was from DAKO (Carpinteria, CA, USA), antibody specific for cleaved caspase-3 (catalogue # 9661) was from Cell Signaling Technology (Beverly, MA, USA), antibody against Oncogene procaspase-9 (catalogue # 556585) was from BD Pharmingen, Palo Alto, CA, USA and the antibody against actin was from Oncogene Research Products (Boston, MA, USA).

Techniques: Western Blot, Control

Journal: Scientific Reports

Article Title: High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis

doi: 10.1038/srep15587

Figure Lengend Snippet: Specific regions of ATRX ( a ), TP53 ( b ) and MIR615 ( c ) genes were targeted using CRISPR/Cas9 system (top) and the efficiency of the sgRNA used were examined via SURVEYOR assay (bottom). Top: The exons/coding region of each gene are represented by blue boxes and are numbered accordingly. CRISPR/Cas9 target sequences are given in green (protospacer) and red (protospacer adjacent motif, PAM) and the corresponding name of the sgRNA are shown in bold. Middle: Grey arrows represent PCR primers used to amplify targeted regions. Red arrows indicate the expected cleavage site of each sgRNA target and the expected sizes of the cleavage product are given next to them. Bottom: SURVEYOR mutation detection assay results for each sgRNA tested. Red stars indicate the cleavage products of the samples indicated above each lane and the numbers at the bottom indicate estimated indel frequency. PC: positive control (G and C control from SURVEYOR assay kit); EV: empty vector (without sgRNA expression cassette).

Article Snippet: The following antibodies were used in our study: rabbit polyclonal anti-ATRX antibody from Santa Cruz Biotechnology (sc-15408); mouse monoclonal anti-p53 antibody from Santa Cruz Biotechnology (sc-126); mouse monoclonal anti-β-actin antibody from Sigma-Aldrich Co. (A1978); rabbit monoclonal anti-Akt2 antibody from Cell Signaling Technology (#3063); sheep anti-mouse HRP-conjugated secondary antibody from Jackson ImmunoResearch; and goat anti-rabbit HRP-conjugated secondary antibody from Jackson ImmunoResearch.

Techniques: CRISPR, Mutagenesis, Detection Assay, Positive Control, Control, Plasmid Preparation, Expressing

Journal: Scientific Reports

Article Title: High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis

doi: 10.1038/srep15587

Figure Lengend Snippet: TP53 -targeted HCT116 cells were genotyped using fluorescent PCR-capillary gel electrophoresis ( a ) and several representative clones are shown. Clones #1 to #3 and clones #4 to #6 were targeted by sgTP53-E4.1 and –E4.2, respectively. ( b ) Sanger sequencing was performed to validate the indel mutations harboured by each clones. Representation of mutations is similar to that in ; in addition, orange nucleotides indicate substituted bases. Quantitative RT-PCR ( c ) and Western blot ( d ) analyses were performed to confirm the genotype of the clones. Error bars represent standard deviations of values from two independent experiments (n = 2). Asterisks represent significantly different (p < 0.05) expression levels as compared to the wildtype parental clone using one-tailed t-test.

Article Snippet: The following antibodies were used in our study: rabbit polyclonal anti-ATRX antibody from Santa Cruz Biotechnology (sc-15408); mouse monoclonal anti-p53 antibody from Santa Cruz Biotechnology (sc-126); mouse monoclonal anti-β-actin antibody from Sigma-Aldrich Co. (A1978); rabbit monoclonal anti-Akt2 antibody from Cell Signaling Technology (#3063); sheep anti-mouse HRP-conjugated secondary antibody from Jackson ImmunoResearch; and goat anti-rabbit HRP-conjugated secondary antibody from Jackson ImmunoResearch.

Techniques: Nucleic Acid Electrophoresis, Clone Assay, Sequencing, Quantitative RT-PCR, Western Blot, Expressing, One-tailed Test