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    Polysciences inc plasmid dna
    MxB inhibits Rev-dependent Gag expression. (A) MxB-Flag and its mutated <t>DNA</t> were transfected into HEK293T cells together with GPV-RRE and Rev. Expression of Gag, MxB and Tubulin in cell lysates were determined by Western blotting. <t>Transfection</t> experiment was also performed with GPV-CTE-x4 and MxB-Flag to examine the effect of MxB on Rev-independent Gag expression. Protein markers (kDa) are shown on the right side of the gels. (B) Levels of viral Gag proteins in the Western blots were quantified with Image J, the results from three independent experiments are presented in the bar graph. (C) Levels of viral RT activity in the culture supernatants were determined to measure the levels of VLPs. The values from the vector controls are arbitrarily set as 100. Results shown are the average of three independent transfection experiments. (D) Fluorescent in situ RNA hybridization to detect subcellular localization of the GPV-RRE RNA in the control and MxB-expressing cells. The cytoplasmic GPV-RRE RNA foci and nuclear GPV-RRE RNA foci per cell were counted, and the results are shown in (E) and (F). P values were calculated with reference to the vector control. Scale bar represents 10 µm. * indicates p
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    1) Product Images from "HIV-1 resists MxB inhibition of viral Rev protein"

    Article Title: HIV-1 resists MxB inhibition of viral Rev protein

    Journal: Emerging Microbes & Infections

    doi: 10.1080/22221751.2020.1818633

    MxB inhibits Rev-dependent Gag expression. (A) MxB-Flag and its mutated DNA were transfected into HEK293T cells together with GPV-RRE and Rev. Expression of Gag, MxB and Tubulin in cell lysates were determined by Western blotting. Transfection experiment was also performed with GPV-CTE-x4 and MxB-Flag to examine the effect of MxB on Rev-independent Gag expression. Protein markers (kDa) are shown on the right side of the gels. (B) Levels of viral Gag proteins in the Western blots were quantified with Image J, the results from three independent experiments are presented in the bar graph. (C) Levels of viral RT activity in the culture supernatants were determined to measure the levels of VLPs. The values from the vector controls are arbitrarily set as 100. Results shown are the average of three independent transfection experiments. (D) Fluorescent in situ RNA hybridization to detect subcellular localization of the GPV-RRE RNA in the control and MxB-expressing cells. The cytoplasmic GPV-RRE RNA foci and nuclear GPV-RRE RNA foci per cell were counted, and the results are shown in (E) and (F). P values were calculated with reference to the vector control. Scale bar represents 10 µm. * indicates p
    Figure Legend Snippet: MxB inhibits Rev-dependent Gag expression. (A) MxB-Flag and its mutated DNA were transfected into HEK293T cells together with GPV-RRE and Rev. Expression of Gag, MxB and Tubulin in cell lysates were determined by Western blotting. Transfection experiment was also performed with GPV-CTE-x4 and MxB-Flag to examine the effect of MxB on Rev-independent Gag expression. Protein markers (kDa) are shown on the right side of the gels. (B) Levels of viral Gag proteins in the Western blots were quantified with Image J, the results from three independent experiments are presented in the bar graph. (C) Levels of viral RT activity in the culture supernatants were determined to measure the levels of VLPs. The values from the vector controls are arbitrarily set as 100. Results shown are the average of three independent transfection experiments. (D) Fluorescent in situ RNA hybridization to detect subcellular localization of the GPV-RRE RNA in the control and MxB-expressing cells. The cytoplasmic GPV-RRE RNA foci and nuclear GPV-RRE RNA foci per cell were counted, and the results are shown in (E) and (F). P values were calculated with reference to the vector control. Scale bar represents 10 µm. * indicates p

    Techniques Used: Expressing, Transfection, Western Blot, Activity Assay, Plasmid Preparation, In Situ, Hybridization

    Effect of MxB on the association of Rev with TNPO1. (A, B) HeLa cells were transfected with Rev together with the GFP vector, MxB-GFP or Δ(1-25) MxB-GFP DNA. The association of Rev with TNPO1 was examined with PLA, and detected as red puncta in cells. Images of one representative experiment are shown in (A). The red puncta per cell from multiple cells of each transfection condition were scored, the results are presented in (B). (C) Western blots of TNPO1 expression in HEK293T or HeLa cells that were stably transduced with Cas9 and sgControl, sgTNPO1-1 or sgTNPO1-2. (D) Subcellular localization of Rev-RFP in HeLa cells with TNPO1 knockdown with Cas9/sgTNPO1. (E) Subcellular localization of Rev in 40 cells of either control or TNPO1 knockdown was determined, and the percentage results are presented in the bar graph. (F) Rev-dependent expression of Gag protein in HEK293T cells that were depleted of TNPO1 with Cas9/sgTNPO1. (G) Levels of Gag in the Western blots were determined with Image J, and the results of three independent experiments are shown in the bar graph. (H) Levels of viral RT activity in the supernatants of transfected HEK293T cells. The RT values from the Cas9/sgControl cells, which were transfected with GPV-RRE and Rev DNA, are arbitrarily set as 100. Results shown are the average of three independent transfection experiments. * indicates p
    Figure Legend Snippet: Effect of MxB on the association of Rev with TNPO1. (A, B) HeLa cells were transfected with Rev together with the GFP vector, MxB-GFP or Δ(1-25) MxB-GFP DNA. The association of Rev with TNPO1 was examined with PLA, and detected as red puncta in cells. Images of one representative experiment are shown in (A). The red puncta per cell from multiple cells of each transfection condition were scored, the results are presented in (B). (C) Western blots of TNPO1 expression in HEK293T or HeLa cells that were stably transduced with Cas9 and sgControl, sgTNPO1-1 or sgTNPO1-2. (D) Subcellular localization of Rev-RFP in HeLa cells with TNPO1 knockdown with Cas9/sgTNPO1. (E) Subcellular localization of Rev in 40 cells of either control or TNPO1 knockdown was determined, and the percentage results are presented in the bar graph. (F) Rev-dependent expression of Gag protein in HEK293T cells that were depleted of TNPO1 with Cas9/sgTNPO1. (G) Levels of Gag in the Western blots were determined with Image J, and the results of three independent experiments are shown in the bar graph. (H) Levels of viral RT activity in the supernatants of transfected HEK293T cells. The RT values from the Cas9/sgControl cells, which were transfected with GPV-RRE and Rev DNA, are arbitrarily set as 100. Results shown are the average of three independent transfection experiments. * indicates p

    Techniques Used: Transfection, Plasmid Preparation, Proximity Ligation Assay, Western Blot, Expressing, Stable Transfection, Transduction, Activity Assay

    2) Product Images from "A non-invasive far-red light-induced split-Cre recombinase system for controllable genome engineering in mice"

    Article Title: A non-invasive far-red light-induced split-Cre recombinase system for controllable genome engineering in mice

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17530-9

    Characterization of the FISC system. a Cre-catalyzed DNA recombination with FISC system using fluorescence imaging and flow cytometry. HEK-293 cells (6 × 10 4 ) were cotransfected with pXY137, pXY169 (P hCMV -CreN59-L9-Coh2-NES-pA), pXY177 (P FRLd -NLS-DocS-L9-CreC60-pA), and pDL78 (P hCMV - loxP -STOP- loxP -EGFP-pA) at a ratio of 10:1:1:20 (w/w/w/w), illuminated for 6 h with FRL (1.5 mW cm -2 , 730 nm) each day for 2 days, and expression of the reporter protein EGFP was visualized by fluorescence microscopy and by flow cytometry at 48 h after the first illumination. Representative images from n = 5 biological replicates. Scale bar, 250 μm. Graph bars represent mean ± SD of n = 3 biological replicates. b Cre-catalyzed DNA recombination with FISC system using luciferase assay. HEK-293 cells (6 × 10 4 ) were cotransfected with pXY137, pXY169, pXY177, and pXY185 (P hCMV - loxP -STOP- loxP -Luciferase-pA) at a ratio of 10:1:1:20 (w/w/w/w), illuminated as described in ( a ), and bioluminescence measurements were taken at 48 h after the first illumination ( n = 3 independent experiments). c Assessment of illumination-intensity-dependent FISC system activity. 6 × 10 4 HEK-293 cells were cotransfected with pXY137, pXY169, pXY177, and pGY125 (SEAP reporter plasmid) at a 10:1:1:20 (w/w/w/w) ratio and illuminated with FRL for 6 h each day for 2 days at eight different light intensities (0–5 mW cm −2 ); SEAP levels were profiled at 48 h after the first illumination ( n = 3 independent experiments). The orange frame marks the highest-fold induction mediated by FISC system. d Exposure-time-dependent FISC system activity. With the same plasmid ratios as in ( c ), we illuminated transfected cells with FRL (1.5 mW cm −2 , 730 nm) for different time periods (0–120 h). SEAP expression was profiled in the cell culture supernatant at 120 h after initial illumination ( n = 3 independent experiments). The orange frame marks the highest-fold induction mediated by FISC system. e FISC-induced SEAP expression in multiple mammalian cell lines. Four different mammalian cell lines were cotransfected and illuminated as described in ( c ), and SEAP expression in the culture supernatant was profiled at 48 h after the first illumination ( n = 3 independent experiments). f Evaluation of the spatial resolution for FISC-mediated transgene expression. A monolayer comprising HEK-293 cells was cotransfected with pXY137, pXY169, pXY177, and pDL78 (EGFP reporter plasmid) at a ratio of a 10:1:1:20 (w/w/w/w), and illuminated as described in ( a ), but through a photomask (schematic, left) with a 6.5 mm slit, and fluorescence microscopy based analysis of the corresponding pattern of EGFP expression at 48 h after the first illumination (right). Representative images from n = 2 biological replicates. b – e Data represent the mean ± SD. Source data for this figure are available in the Source data file.
    Figure Legend Snippet: Characterization of the FISC system. a Cre-catalyzed DNA recombination with FISC system using fluorescence imaging and flow cytometry. HEK-293 cells (6 × 10 4 ) were cotransfected with pXY137, pXY169 (P hCMV -CreN59-L9-Coh2-NES-pA), pXY177 (P FRLd -NLS-DocS-L9-CreC60-pA), and pDL78 (P hCMV - loxP -STOP- loxP -EGFP-pA) at a ratio of 10:1:1:20 (w/w/w/w), illuminated for 6 h with FRL (1.5 mW cm -2 , 730 nm) each day for 2 days, and expression of the reporter protein EGFP was visualized by fluorescence microscopy and by flow cytometry at 48 h after the first illumination. Representative images from n = 5 biological replicates. Scale bar, 250 μm. Graph bars represent mean ± SD of n = 3 biological replicates. b Cre-catalyzed DNA recombination with FISC system using luciferase assay. HEK-293 cells (6 × 10 4 ) were cotransfected with pXY137, pXY169, pXY177, and pXY185 (P hCMV - loxP -STOP- loxP -Luciferase-pA) at a ratio of 10:1:1:20 (w/w/w/w), illuminated as described in ( a ), and bioluminescence measurements were taken at 48 h after the first illumination ( n = 3 independent experiments). c Assessment of illumination-intensity-dependent FISC system activity. 6 × 10 4 HEK-293 cells were cotransfected with pXY137, pXY169, pXY177, and pGY125 (SEAP reporter plasmid) at a 10:1:1:20 (w/w/w/w) ratio and illuminated with FRL for 6 h each day for 2 days at eight different light intensities (0–5 mW cm −2 ); SEAP levels were profiled at 48 h after the first illumination ( n = 3 independent experiments). The orange frame marks the highest-fold induction mediated by FISC system. d Exposure-time-dependent FISC system activity. With the same plasmid ratios as in ( c ), we illuminated transfected cells with FRL (1.5 mW cm −2 , 730 nm) for different time periods (0–120 h). SEAP expression was profiled in the cell culture supernatant at 120 h after initial illumination ( n = 3 independent experiments). The orange frame marks the highest-fold induction mediated by FISC system. e FISC-induced SEAP expression in multiple mammalian cell lines. Four different mammalian cell lines were cotransfected and illuminated as described in ( c ), and SEAP expression in the culture supernatant was profiled at 48 h after the first illumination ( n = 3 independent experiments). f Evaluation of the spatial resolution for FISC-mediated transgene expression. A monolayer comprising HEK-293 cells was cotransfected with pXY137, pXY169, pXY177, and pDL78 (EGFP reporter plasmid) at a ratio of a 10:1:1:20 (w/w/w/w), and illuminated as described in ( a ), but through a photomask (schematic, left) with a 6.5 mm slit, and fluorescence microscopy based analysis of the corresponding pattern of EGFP expression at 48 h after the first illumination (right). Representative images from n = 2 biological replicates. b – e Data represent the mean ± SD. Source data for this figure are available in the Source data file.

    Techniques Used: Fluorescence, Imaging, Flow Cytometry, Expressing, Microscopy, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Cell Culture

    AAV delivery of FISC-mediated DNA recombination in transgenic Cre-tdTomato reporter mice. a Schematic depicting the genetic configuration AAV vectors for the FISC DNA recombination system. b Schematic of working principle for transgenic Cre-tdTomato reporter mice in which the loxP -flanked STOP cassette can be excised by Cre recombinase to allow Cre reporter (tdTomato) expression. c Schematic representation of the experimental procedure for FISC-mediated DNA recombination activity in mice. d Representative images of the tdTomato fluorescence in the AAV-transducted reporter mice ( n = 3 mice/group). e The fluorescence measurements of the tdTomato expression shown in ( d ) ( n = 3 mice/group). f Representative images of the tdTomato fluorescence in isolated liver tissue from the AAV-transducted mice shown in ( d ) ( n = 3 mice/group). Scale bar, 1 cm. g Bioluminescence tdTomato expression shown in ( f ) ( n = 3 mice/group). h , i qRT-PCR ( h ) and Western blot ( i ) analysis of tdTomato in isolated liver tissue shown in ( f ) ( n = 3 mice/group). Western blotting images are representative of three mice from two independent experiments. j Representative fluorescence images of the liver sections of the transgenic Cre-tdTomato reporter mice shown in ( f ) ( n = 5 biological replicates. Scale bar, 250 μm). Data in ( e , g , h ) represent the mean ± SEM. Source data for this figure are available in the Source data file.
    Figure Legend Snippet: AAV delivery of FISC-mediated DNA recombination in transgenic Cre-tdTomato reporter mice. a Schematic depicting the genetic configuration AAV vectors for the FISC DNA recombination system. b Schematic of working principle for transgenic Cre-tdTomato reporter mice in which the loxP -flanked STOP cassette can be excised by Cre recombinase to allow Cre reporter (tdTomato) expression. c Schematic representation of the experimental procedure for FISC-mediated DNA recombination activity in mice. d Representative images of the tdTomato fluorescence in the AAV-transducted reporter mice ( n = 3 mice/group). e The fluorescence measurements of the tdTomato expression shown in ( d ) ( n = 3 mice/group). f Representative images of the tdTomato fluorescence in isolated liver tissue from the AAV-transducted mice shown in ( d ) ( n = 3 mice/group). Scale bar, 1 cm. g Bioluminescence tdTomato expression shown in ( f ) ( n = 3 mice/group). h , i qRT-PCR ( h ) and Western blot ( i ) analysis of tdTomato in isolated liver tissue shown in ( f ) ( n = 3 mice/group). Western blotting images are representative of three mice from two independent experiments. j Representative fluorescence images of the liver sections of the transgenic Cre-tdTomato reporter mice shown in ( f ) ( n = 5 biological replicates. Scale bar, 250 μm). Data in ( e , g , h ) represent the mean ± SEM. Source data for this figure are available in the Source data file.

    Techniques Used: Transgenic Assay, Mouse Assay, Expressing, Activity Assay, Fluorescence, Isolation, Quantitative RT-PCR, Western Blot

    FISC-induced DNA recombination in BALB/c wild-type mice. a Schematic showing the experimental procedure of light-induced DNA recombination activity in mice. b Comparison of the FISC system with the CRY2-Cre and PA-Cre systems using in vivo bioluminescence imaging. The BALB/c mice were transiently hydrodynamically injected (tail vein) with an iteration of the FISC system comprising three plasmids [pXY137/pXY237 (pA-CreC60-DocS-NLS-P FRLd -Space3-P hCMV -CreN59-Coh2-P2A-ZeoR-pA)/pXY185 (luciferase reporter plasmid) at a ratio of 1.5:1:1 (w/w/w)], or the CRY2-Cre, or the PA-Cre system or only the luciferase reporter plasmid pXY185 as control. At 8 h after the injection, the mice were illuminated with FRL (20 mW cm −2 , 730 nm) or blue light (BL, 20 mW cm −2 , 460 nm) for 12 h (15 min on, 15 min off, alternating) or maintained in the dark, followed by imaging at 12 h after light illumination. c Bioluminescence measurements of the BALB/c mice shown in ( b ) (Control: n = 3, FISC system: n = 4, CRY2-Cre and PA-Cre: n = 5. Data present the mean ± SEM). See Supplementary Table 4 for detailed description of genetic components for each optogenetic system. Source data for this figure are available in the Source data file.
    Figure Legend Snippet: FISC-induced DNA recombination in BALB/c wild-type mice. a Schematic showing the experimental procedure of light-induced DNA recombination activity in mice. b Comparison of the FISC system with the CRY2-Cre and PA-Cre systems using in vivo bioluminescence imaging. The BALB/c mice were transiently hydrodynamically injected (tail vein) with an iteration of the FISC system comprising three plasmids [pXY137/pXY237 (pA-CreC60-DocS-NLS-P FRLd -Space3-P hCMV -CreN59-Coh2-P2A-ZeoR-pA)/pXY185 (luciferase reporter plasmid) at a ratio of 1.5:1:1 (w/w/w)], or the CRY2-Cre, or the PA-Cre system or only the luciferase reporter plasmid pXY185 as control. At 8 h after the injection, the mice were illuminated with FRL (20 mW cm −2 , 730 nm) or blue light (BL, 20 mW cm −2 , 460 nm) for 12 h (15 min on, 15 min off, alternating) or maintained in the dark, followed by imaging at 12 h after light illumination. c Bioluminescence measurements of the BALB/c mice shown in ( b ) (Control: n = 3, FISC system: n = 4, CRY2-Cre and PA-Cre: n = 5. Data present the mean ± SEM). See Supplementary Table 4 for detailed description of genetic components for each optogenetic system. Source data for this figure are available in the Source data file.

    Techniques Used: Mouse Assay, Activity Assay, In Vivo, Imaging, Injection, Luciferase, Plasmid Preparation

    Design and optimization of the far-red light-induced split Cre- loxP system (FISC system). a Schematic representation of the FISC system. Cre recombinase was split into two fragments: CreN59 (residues 1–59) fused to Coh2 driven by a constitutive promoter (P hCMV ) and CreC60 (residues 60–343) fused to DocS driven by the far-red light (FRL, 730 nm)-inducible promoter (P FRLx ). Upon FRL illumination, the photoreceptor BphS is activated to convert intracellular guanylate triphosphate (GTP) into cyclic diguanylate monophosphate (c-di-GMP). The cytosolic c-di-GMP production induces binding of the far-red light-dependent transactivator FRTA (p65-VP64-BldD) to its synthetic promoter P FRLx to drive DocS-CreC60 expression. Consequently, the catalytic activities of Cre recombinase can be restored once the two Cre fragments assemble based on affinity interactions of their respective Coh2 and DocS fusion domains, enabling to excise DNA sequences flanked by loxP sites. b Schematic depicting the genetic configuration of constructs used in the FISC system. pA, polyadenylation signals; YhjH, the bacterial c-di-GMP phosphodiesterase; DocS, dockerin S from C. thermocellum complexed with Coh2; L 1-11 , different linkers from 1 to 11 (Supplementary Table 1 ), Coh2, an anchoring protein from C. thermocellum ; loxP , the specific Cre recombinase binding site; STOP, a terminator containing pA to prevent transcription; GOI, gene of interest. c Schematic depicting different genetic configurations of the FRL-inducible promoters P FRLx . 3*whiG, three copies of BldD-specific binding sequence; P hCMVmin , minimal version of P hCMV ; P min , minimal eukaryotic promoter; TATA, minimal eukaryotic promoter with only TATA box. d Schematic depicting the time schedule for the FISC experimental procedure with mammalian cells. e Optimization of the different transfection amounts for CreN59-Coh2 expression and light-inducible P FRLd -driven DocS-CreC60 expression. HEK-293 (6 × 10 4 ) cells were cotransfected with pXY137 (P hCMV -p65-VP64-BldD-pA::P hCMV -BphS-P2A-YhjH-pA, 100 ng), pGY125 (P hCMV - loxP -STOP- loxP -SEAP-pA, 200 ng), pXY110 (P hCMV -CreN59-L0-Coh2-NES-pA) and pXY133 (P FRLd -NLS-DocS-L0-CreC60-pA) from 5 to 100 ng at a ratio of 1:1 (w/w), and then illuminated for 6 h with FRL (1.5 mW cm -2 , 730 nm) once each day for 2 days. SEAP expression in the culture supernatants was profiled at 48 h after the first illumination ( n = 3 independent experiments). f Optimization of the different linkers (L1–L11) between the CreN59 and Coh2 domains, as well as the CreC60 and DocS domains. The 6 × 10 4 HEK-293 cells per well were cotransfected with pXY137 (100 ng), pGY125 (200 ng), and Docs-CreC60 and Coh2-CreN59 with different combinations of the linkers (10 ng/10 ng) (Supplementary Table 2 ); these were illuminated as described in e , followed by SEAP expression in the culture supernatants profiled at 48 h after the first illumination ( n = 3 independent experiments). The orange frame in ( e , f ) marks the best-performing condition. e , f Data represent the mean ± SD. Source data for this figure are available in the Source data file.
    Figure Legend Snippet: Design and optimization of the far-red light-induced split Cre- loxP system (FISC system). a Schematic representation of the FISC system. Cre recombinase was split into two fragments: CreN59 (residues 1–59) fused to Coh2 driven by a constitutive promoter (P hCMV ) and CreC60 (residues 60–343) fused to DocS driven by the far-red light (FRL, 730 nm)-inducible promoter (P FRLx ). Upon FRL illumination, the photoreceptor BphS is activated to convert intracellular guanylate triphosphate (GTP) into cyclic diguanylate monophosphate (c-di-GMP). The cytosolic c-di-GMP production induces binding of the far-red light-dependent transactivator FRTA (p65-VP64-BldD) to its synthetic promoter P FRLx to drive DocS-CreC60 expression. Consequently, the catalytic activities of Cre recombinase can be restored once the two Cre fragments assemble based on affinity interactions of their respective Coh2 and DocS fusion domains, enabling to excise DNA sequences flanked by loxP sites. b Schematic depicting the genetic configuration of constructs used in the FISC system. pA, polyadenylation signals; YhjH, the bacterial c-di-GMP phosphodiesterase; DocS, dockerin S from C. thermocellum complexed with Coh2; L 1-11 , different linkers from 1 to 11 (Supplementary Table 1 ), Coh2, an anchoring protein from C. thermocellum ; loxP , the specific Cre recombinase binding site; STOP, a terminator containing pA to prevent transcription; GOI, gene of interest. c Schematic depicting different genetic configurations of the FRL-inducible promoters P FRLx . 3*whiG, three copies of BldD-specific binding sequence; P hCMVmin , minimal version of P hCMV ; P min , minimal eukaryotic promoter; TATA, minimal eukaryotic promoter with only TATA box. d Schematic depicting the time schedule for the FISC experimental procedure with mammalian cells. e Optimization of the different transfection amounts for CreN59-Coh2 expression and light-inducible P FRLd -driven DocS-CreC60 expression. HEK-293 (6 × 10 4 ) cells were cotransfected with pXY137 (P hCMV -p65-VP64-BldD-pA::P hCMV -BphS-P2A-YhjH-pA, 100 ng), pGY125 (P hCMV - loxP -STOP- loxP -SEAP-pA, 200 ng), pXY110 (P hCMV -CreN59-L0-Coh2-NES-pA) and pXY133 (P FRLd -NLS-DocS-L0-CreC60-pA) from 5 to 100 ng at a ratio of 1:1 (w/w), and then illuminated for 6 h with FRL (1.5 mW cm -2 , 730 nm) once each day for 2 days. SEAP expression in the culture supernatants was profiled at 48 h after the first illumination ( n = 3 independent experiments). f Optimization of the different linkers (L1–L11) between the CreN59 and Coh2 domains, as well as the CreC60 and DocS domains. The 6 × 10 4 HEK-293 cells per well were cotransfected with pXY137 (100 ng), pGY125 (200 ng), and Docs-CreC60 and Coh2-CreN59 with different combinations of the linkers (10 ng/10 ng) (Supplementary Table 2 ); these were illuminated as described in e , followed by SEAP expression in the culture supernatants profiled at 48 h after the first illumination ( n = 3 independent experiments). The orange frame in ( e , f ) marks the best-performing condition. e , f Data represent the mean ± SD. Source data for this figure are available in the Source data file.

    Techniques Used: Binding Assay, Expressing, Construct, Sequencing, Transfection

    3) Product Images from "Familial Amyotrophic Lateral Sclerosis-linked Mutations in Profilin 1 Exacerbate TDP-43-induced Degeneration in the Retina of Drosophila melanogaster through an Increase in the Cytoplasmic Localization of TDP-43 *"

    Article Title: Familial Amyotrophic Lateral Sclerosis-linked Mutations in Profilin 1 Exacerbate TDP-43-induced Degeneration in the Retina of Drosophila melanogaster through an Increase in the Cytoplasmic Localization of TDP-43 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.729152

    Overexpression of fALS mutant PFN1 , but not wt PFN1 , suppressed function of TDP-43. A, semi-quantitative RT-PCR analyses of the splicing pattern of MADD gene in control siRNA-treated, TDP-43 siRNA-treated, EGFP , or myc-tagged wt, C71G, or M114T mutant PFN1 stably expressed HEK293 cells. 200-Base pair DNA size marker was indicated at the left of the panel. B, quantitative ratio of exon 31 skipping of MADD gene. n = 6, mean ± S.E., *, p
    Figure Legend Snippet: Overexpression of fALS mutant PFN1 , but not wt PFN1 , suppressed function of TDP-43. A, semi-quantitative RT-PCR analyses of the splicing pattern of MADD gene in control siRNA-treated, TDP-43 siRNA-treated, EGFP , or myc-tagged wt, C71G, or M114T mutant PFN1 stably expressed HEK293 cells. 200-Base pair DNA size marker was indicated at the left of the panel. B, quantitative ratio of exon 31 skipping of MADD gene. n = 6, mean ± S.E., *, p

    Techniques Used: Over Expression, Mutagenesis, Quantitative RT-PCR, Stable Transfection, Marker

    4) Product Images from "ZC3H12B/MCPIP2, a new active member of the ZC3H12 family"

    Article Title: ZC3H12B/MCPIP2, a new active member of the ZC3H12 family

    Journal: RNA

    doi: 10.1261/rna.071381.119

    ZC3H12B is a cytoplasm-localized negative regulator of cell count. ( A ) ( Upper and middle panels) HeLa cells were transfected with vectors encoding the ZC3H12B protein (containing an amino-terminal V5 tag). After 24 h the cells were fixed and stained using antibodies against the V5 tag (green). The DNA was counterstained using DAPI (blue). Scale bar, 10 µm. ( Lower panel) Zoom-in of the cell presented in the middle panel showing frontal (xz) and transverse (yz) sections revealing the exclusion of the ZC3H12B protein from the nucleus. Scale bars, 5 µm. ( B ) 293T cells were transfected with vectors encoding ZC3H12B proteins (wild-type or the D196A mutein; ZC3H12B WT and ZC3H12B D196A, respectively) or empty control plasmids (empty vector) or left untreated. Every 24 h after transfection (up to 72 h posttransfection), the cells were counted. The graph shows the mean results of three independent experiments ±SD. The data were analyzed using two-way ANOVA with Bonferroni's post-hoc test ([*] P
    Figure Legend Snippet: ZC3H12B is a cytoplasm-localized negative regulator of cell count. ( A ) ( Upper and middle panels) HeLa cells were transfected with vectors encoding the ZC3H12B protein (containing an amino-terminal V5 tag). After 24 h the cells were fixed and stained using antibodies against the V5 tag (green). The DNA was counterstained using DAPI (blue). Scale bar, 10 µm. ( Lower panel) Zoom-in of the cell presented in the middle panel showing frontal (xz) and transverse (yz) sections revealing the exclusion of the ZC3H12B protein from the nucleus. Scale bars, 5 µm. ( B ) 293T cells were transfected with vectors encoding ZC3H12B proteins (wild-type or the D196A mutein; ZC3H12B WT and ZC3H12B D196A, respectively) or empty control plasmids (empty vector) or left untreated. Every 24 h after transfection (up to 72 h posttransfection), the cells were counted. The graph shows the mean results of three independent experiments ±SD. The data were analyzed using two-way ANOVA with Bonferroni's post-hoc test ([*] P

    Techniques Used: Cell Counting, Transfection, Staining, Plasmid Preparation

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    Article Snippet: 2.1 Cell culture and transfection Human embryonic kidney HEK‐293 T cells were cultured in DMEM high glucose supplemented with 10% (v/v) fetal bovine serum, 2 mM L‐glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin at 37°C in 5% CO2 –95% air atmosphere. .. For transfection experiments, cells were grown on 10 mm petri dishes and were incubated for a period of 36 hr with a mixture of 20 μL of 1 mg/ml polyethylenimine (Polysciences) and 5–10 μg of the respective plasmid DNA (KCNJ10, Clc‐kb, and barttin from Origene), as described (Durocher, Perret, & Kamen, ). .. Depending on whether the Vm or [Cl− ]i was going to be measured in the cells, the plasmid constructs pcDNA3.1 (−) VSFP3.1 (Addgene) or pcDNA3.1 (+) EYFP H148Q/I152L (Addgene) were transfected into the cells.

    Article Title: Expression of antibodies using single open reading frame (sORF) vector design
    Article Snippet: Expression in HEK293–6E cells Expression vectors were introduced into HEK 293 cells as previously described. .. Briefly, cells in exponential growth phase were transfected with 0.25–1.0 μg/mL plasmid DNA by adding a complex solution of plasmid DNA and PEI (polyethylenimine linear, 25 kDa) (Polysciences, cat.# 24765–2) at a ratio 1:2. .. After 20–24 h, Tryptone N1 medium (Tekni-Science, cat.# 19553) was added to a final concentration of 0.5%.

    Article Title: Signals in APOBEC3F N-terminal and C-terminal Deaminase Domains Each Contribute to Encapsidation in HIV-1 Virions and Are Both Required for HIV-1 Restriction *
    Article Snippet: The effect on inhibition of virion infectivity of the combined A3F mutations W126A and L306A was evaluated using Compusyn software following the methods described by Chou ( ). .. HEK293T cells were plated at a density of 6 × 105 cells/well in a 6-well culture plate 24 h prior to transfection with either 0.2 μg of pTT5-GST or 1 μg of pTT5-GST-NC and 1 μg of pcDNA-A3F, pcDNA-A3F(W126A), or pcDNA-A3F(L306A) plasmid DNA using linear polyethylenimine (PEI; 25 kDa; Polysciences, Inc.) as described ( ). .. Forty-eight hours after transfection, cells were lysed in 250 μl of cell lysis buffer ((1× Dulbecco's Phosphate-buffered Saline (Mediatech, Inc.), 1 m m Na2 EDTA, 0.5% Triton X-100 (v/v), and complete mini protease inhibitor mixture without Na2 EDTA (Roche)) and centrifuged at 10,000 × g for 5 min at 4 °C.

    Article Title: Lysosome-Associated Membrane Proteins Support the Furin-Mediated Processing of the Mumps Virus Fusion Protein
    Article Snippet: LoVo cells, obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition, were maintained in Ham’s F-12 with l-glutamine (Nacalai) supplemented with 20% (vol/vol) FBS and penicillin/streptomycin. .. Cells were transfected with indicated amounts of plasmid DNAs using Polyethylenimine Max (Polysciences) (293T, HEK293, Vero, and HeLa cells), Lipofectamine LTX (Invitrogen) (A549, H358, and U-87 cells), or Lipofectamine 2000 (Invitrogen) (LoVo cells). .. The GFP-expressing recombinant MuV of the Hoshino strain was recovered from the full-length cDNA plasmid as described previously ( ).

    Article Title: A TRAVELING WAVE ION MOBILITY SPECTROMETRY (TWIMS) STUDY OF THE ROBO1-HEPARAN SULFATE INTERACTION
    Article Snippet: Both cell lines were maintained in culture media comprised of 9 volumes FreeStyleTM 293 expression media (Thermo Fisher Scientific) and 1 volume EX-CELL® 293 serum-free medium (Sigma) (9:1 media) as previously described. .. Suspension cultures (1 L) of either HEK293S (GnTI−) cells or FreeStyleTM 293-F cells were transfected with the respective plasmid DNAs using polyethyleneimine (linear 25 kDa PEI, Polysciences) as transfection reagent as previously described [ ]. .. The cultures were diluted 1:1 with culture medium containing 4.4 mM valproic acid (2.2 mM final concentration) 24 h after transfection, and protein production was continued for a further 5 days at 37°C.

    Incubation:

    Article Title: Modified HEK cells simulate DCT cells in their sensitivity and response to changes in extracellular K. Modified HEK cells simulate DCT cells in their sensitivity and response to changes in extracellular K
    Article Snippet: 2.1 Cell culture and transfection Human embryonic kidney HEK‐293 T cells were cultured in DMEM high glucose supplemented with 10% (v/v) fetal bovine serum, 2 mM L‐glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin at 37°C in 5% CO2 –95% air atmosphere. .. For transfection experiments, cells were grown on 10 mm petri dishes and were incubated for a period of 36 hr with a mixture of 20 μL of 1 mg/ml polyethylenimine (Polysciences) and 5–10 μg of the respective plasmid DNA (KCNJ10, Clc‐kb, and barttin from Origene), as described (Durocher, Perret, & Kamen, ). .. Depending on whether the Vm or [Cl− ]i was going to be measured in the cells, the plasmid constructs pcDNA3.1 (−) VSFP3.1 (Addgene) or pcDNA3.1 (+) EYFP H148Q/I152L (Addgene) were transfected into the cells.

    Plasmid Preparation:

    Article Title: Modified HEK cells simulate DCT cells in their sensitivity and response to changes in extracellular K. Modified HEK cells simulate DCT cells in their sensitivity and response to changes in extracellular K
    Article Snippet: 2.1 Cell culture and transfection Human embryonic kidney HEK‐293 T cells were cultured in DMEM high glucose supplemented with 10% (v/v) fetal bovine serum, 2 mM L‐glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin at 37°C in 5% CO2 –95% air atmosphere. .. For transfection experiments, cells were grown on 10 mm petri dishes and were incubated for a period of 36 hr with a mixture of 20 μL of 1 mg/ml polyethylenimine (Polysciences) and 5–10 μg of the respective plasmid DNA (KCNJ10, Clc‐kb, and barttin from Origene), as described (Durocher, Perret, & Kamen, ). .. Depending on whether the Vm or [Cl− ]i was going to be measured in the cells, the plasmid constructs pcDNA3.1 (−) VSFP3.1 (Addgene) or pcDNA3.1 (+) EYFP H148Q/I152L (Addgene) were transfected into the cells.

    Article Title: Expression of antibodies using single open reading frame (sORF) vector design
    Article Snippet: Expression in HEK293–6E cells Expression vectors were introduced into HEK 293 cells as previously described. .. Briefly, cells in exponential growth phase were transfected with 0.25–1.0 μg/mL plasmid DNA by adding a complex solution of plasmid DNA and PEI (polyethylenimine linear, 25 kDa) (Polysciences, cat.# 24765–2) at a ratio 1:2. .. After 20–24 h, Tryptone N1 medium (Tekni-Science, cat.# 19553) was added to a final concentration of 0.5%.

    Article Title: Antibody-based delivery of Interleukin-9 to neovascular structures: therapeutic evaluation in cancer and arthritis
    Article Snippet: For transfection 4 × 106 cells/mL cells were resuspended in ProCHO-4 Medium (Lonza) supplemented with 4 mM Ultraglutamine (Lonza). .. 0.625 μg of plasmid DNAs per million cells followed by 2.5 μg polyethyleneimine per million cells (Polysciences) were added to the cells and gently mixed. .. Transfected cultures were incubated in a shaking incubator at 31°C with 5% CO2 atmosphere shaking at 120 rpm for 6 days.

    Article Title: Signals in APOBEC3F N-terminal and C-terminal Deaminase Domains Each Contribute to Encapsidation in HIV-1 Virions and Are Both Required for HIV-1 Restriction *
    Article Snippet: The effect on inhibition of virion infectivity of the combined A3F mutations W126A and L306A was evaluated using Compusyn software following the methods described by Chou ( ). .. HEK293T cells were plated at a density of 6 × 105 cells/well in a 6-well culture plate 24 h prior to transfection with either 0.2 μg of pTT5-GST or 1 μg of pTT5-GST-NC and 1 μg of pcDNA-A3F, pcDNA-A3F(W126A), or pcDNA-A3F(L306A) plasmid DNA using linear polyethylenimine (PEI; 25 kDa; Polysciences, Inc.) as described ( ). .. Forty-eight hours after transfection, cells were lysed in 250 μl of cell lysis buffer ((1× Dulbecco's Phosphate-buffered Saline (Mediatech, Inc.), 1 m m Na2 EDTA, 0.5% Triton X-100 (v/v), and complete mini protease inhibitor mixture without Na2 EDTA (Roche)) and centrifuged at 10,000 × g for 5 min at 4 °C.

    Article Title: Lysosome-Associated Membrane Proteins Support the Furin-Mediated Processing of the Mumps Virus Fusion Protein
    Article Snippet: LoVo cells, obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition, were maintained in Ham’s F-12 with l-glutamine (Nacalai) supplemented with 20% (vol/vol) FBS and penicillin/streptomycin. .. Cells were transfected with indicated amounts of plasmid DNAs using Polyethylenimine Max (Polysciences) (293T, HEK293, Vero, and HeLa cells), Lipofectamine LTX (Invitrogen) (A549, H358, and U-87 cells), or Lipofectamine 2000 (Invitrogen) (LoVo cells). .. The GFP-expressing recombinant MuV of the Hoshino strain was recovered from the full-length cDNA plasmid as described previously ( ).

    Article Title: A TRAVELING WAVE ION MOBILITY SPECTROMETRY (TWIMS) STUDY OF THE ROBO1-HEPARAN SULFATE INTERACTION
    Article Snippet: Both cell lines were maintained in culture media comprised of 9 volumes FreeStyleTM 293 expression media (Thermo Fisher Scientific) and 1 volume EX-CELL® 293 serum-free medium (Sigma) (9:1 media) as previously described. .. Suspension cultures (1 L) of either HEK293S (GnTI−) cells or FreeStyleTM 293-F cells were transfected with the respective plasmid DNAs using polyethyleneimine (linear 25 kDa PEI, Polysciences) as transfection reagent as previously described [ ]. .. The cultures were diluted 1:1 with culture medium containing 4.4 mM valproic acid (2.2 mM final concentration) 24 h after transfection, and protein production was continued for a further 5 days at 37°C.

    Article Title: Simple paired heavy- and light-chain antibody repertoire sequencing using endoplasmic reticulum microsomes
    Article Snippet: HEK 293 T cells were cultured using rich glucose (4.5 g/L D-glucose) Dulbecco’s Modified Eagle’s Medium (Gibco BRL) supplemented with heat-inactivated ultra-low IgG fetal bovine serum (Thermo Fisher Scientific), 100 U/mL penicillin, and 100 μg/mL streptomycin. .. Purified plasmid DNAs for paired HC and LC clonotypes were co-transfected into 85–95% confluent HEK 293 T cells using PEI (polyethyleneamine, Polysciences). .. Culture supernatants were collected four days after transfection and TT antigen-specific clonotypes were identified by indirect ELISA.

    Article Title: Sortase-Mediated Site-Specific Modification of Interleukin-2 for the Generation of a Tumor-Targeting Acetazolamide–Cytokine Conjugate
    Article Snippet: For 1 mL of production, 4 × 106 cells were collected by centrifugation and resuspended in ProCHO4 media (Lonza) supplemented with 4 mM Ultraglutamine (Lonza). .. Per million cells, 0.625 μg of plasmid DNAs followed by 2.5 μg of polyethylenimine (1 mg/mL solution in water at pH 7.0, Polysciences) were added to the cells and gently mixed. ..

    Purification:

    Article Title: Simple paired heavy- and light-chain antibody repertoire sequencing using endoplasmic reticulum microsomes
    Article Snippet: HEK 293 T cells were cultured using rich glucose (4.5 g/L D-glucose) Dulbecco’s Modified Eagle’s Medium (Gibco BRL) supplemented with heat-inactivated ultra-low IgG fetal bovine serum (Thermo Fisher Scientific), 100 U/mL penicillin, and 100 μg/mL streptomycin. .. Purified plasmid DNAs for paired HC and LC clonotypes were co-transfected into 85–95% confluent HEK 293 T cells using PEI (polyethyleneamine, Polysciences). .. Culture supernatants were collected four days after transfection and TT antigen-specific clonotypes were identified by indirect ELISA.

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    Polysciences inc plasmid dna
    EZH2 directly methylates REST (A) Immunoprecipitation analysis of HAFs with anti-IgG or anti-EZH2 antibody followed by immunoblot analysis with anti-REST antibody. (B) Immunoprecipitation analysis of HAFs treated with DMSO or GSK126 (3 uM, 48 hr) using anti-pan-methyl-lysine antibody followed by immunoblot analysis with anti-REST antibody. (C) Immunoprecipitation analysis of HEK293T cells <t>co-transfected</t> with REST and EZH2 (left) or shEZH2 (right) using anti-pan-methyl-lysine antibody followed by immunoblot analysis with anti-REST antibody. (D) The methylation site, K494 of REST within lysine-rich domain and sequence comparison between methylation sites of Histone3/RORα and K494 of REST in human, mouse, and rat. RD, repression domain; DBD, <t>DNA</t> binding domain; K-rich, Lysine rich domain; P-rich, Proline rich domain. (E) Immunoprecipitation analysis of HEK293T cells co-transfected with Flag-REST-WT or K494A using anti-Flag antibody followed by immunoblot analysis with anti-pan-methyl-lysine antibody and anti-Flag antibody.
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    EZH2 directly methylates REST (A) Immunoprecipitation analysis of HAFs with anti-IgG or anti-EZH2 antibody followed by immunoblot analysis with anti-REST antibody. (B) Immunoprecipitation analysis of HAFs treated with DMSO or GSK126 (3 uM, 48 hr) using anti-pan-methyl-lysine antibody followed by immunoblot analysis with anti-REST antibody. (C) Immunoprecipitation analysis of HEK293T cells co-transfected with REST and EZH2 (left) or shEZH2 (right) using anti-pan-methyl-lysine antibody followed by immunoblot analysis with anti-REST antibody. (D) The methylation site, K494 of REST within lysine-rich domain and sequence comparison between methylation sites of Histone3/RORα and K494 of REST in human, mouse, and rat. RD, repression domain; DBD, DNA binding domain; K-rich, Lysine rich domain; P-rich, Proline rich domain. (E) Immunoprecipitation analysis of HEK293T cells co-transfected with Flag-REST-WT or K494A using anti-Flag antibody followed by immunoblot analysis with anti-pan-methyl-lysine antibody and anti-Flag antibody.

    Journal: Developmental cell

    Article Title: MicroRNAs Overcome Cell Fate Barrier by Reducing EZH2-Controlled REST Stability during Neuronal Conversion of Human Adult Fibroblasts

    doi: 10.1016/j.devcel.2018.06.007

    Figure Lengend Snippet: EZH2 directly methylates REST (A) Immunoprecipitation analysis of HAFs with anti-IgG or anti-EZH2 antibody followed by immunoblot analysis with anti-REST antibody. (B) Immunoprecipitation analysis of HAFs treated with DMSO or GSK126 (3 uM, 48 hr) using anti-pan-methyl-lysine antibody followed by immunoblot analysis with anti-REST antibody. (C) Immunoprecipitation analysis of HEK293T cells co-transfected with REST and EZH2 (left) or shEZH2 (right) using anti-pan-methyl-lysine antibody followed by immunoblot analysis with anti-REST antibody. (D) The methylation site, K494 of REST within lysine-rich domain and sequence comparison between methylation sites of Histone3/RORα and K494 of REST in human, mouse, and rat. RD, repression domain; DBD, DNA binding domain; K-rich, Lysine rich domain; P-rich, Proline rich domain. (E) Immunoprecipitation analysis of HEK293T cells co-transfected with Flag-REST-WT or K494A using anti-Flag antibody followed by immunoblot analysis with anti-pan-methyl-lysine antibody and anti-Flag antibody.

    Article Snippet: The cells were transfected in triplicate with 100 ng plasmid DNA each per well using PEI (Polyethylenimine) (Polysciences) according to the manufactures instructions.

    Techniques: Immunoprecipitation, Transfection, Methylation, Sequencing, Binding Assay

    ATM and PAXX deficiency accentuates the c-NHEJ deficits of L115A. (A) Zeocin sensitivity of parental wild-type cells, XLF-deficient and XLF −/− PAXX −/− 293 cells (left), and HCT116 cells (right). Error bars indicate SEM from three independent experiments. (B) Recombination percentage of episomal fluorescent coding and signal joining substrates comparing XLF-deficient, XLF/ATM doubly deficient, and XLF −/− PAXX −/− 293 cells transiently expressing Rag1, Rag2, and wild-type and mutant forms of XLF as indicated. Error bars indicate SEM from at least 3 independent experiments. Note that XLF L115A shows a significantly lower coding end joining rate in XLF-deficient ( P = 0.0001) and XLF −/− ATM −/− ( P = 0.0022) 293 cells, unlike XLF −/− PAXX −/− cells. Similarly, XLF L115A performs significantly reduced signal joining in XLF-deficient ( P = 0.0210) and XLF −/− ATM −/− ( P = 0.0022) cells, unlike XLF −/− PAXX −/− 293 cells. (C) Zeocin sensitivity of the XLF −/− ATM −/− 293 cell strain stably expressing equivalent levels of wt or mutant XLF. Data from three clones expressing L115A are presented. Error bars indicate SEM from three independent experiments. (D) Immunoblot analysis depicting XLF expression levels in XLF −/− ATM −/− cells stably transfected with wt or mutant XLF or vector control. (E) Immunoblot showing levels of DNA-PKcs autophosphorylation at S2056 with and without zeocin (Zeo) treatment of XLF/ATM doubly deficient cells stably expressing wt and mutant forms of XLF.

    Journal: Molecular and Cellular Biology

    Article Title: XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation

    doi: 10.1128/MCB.01503-14

    Figure Lengend Snippet: ATM and PAXX deficiency accentuates the c-NHEJ deficits of L115A. (A) Zeocin sensitivity of parental wild-type cells, XLF-deficient and XLF −/− PAXX −/− 293 cells (left), and HCT116 cells (right). Error bars indicate SEM from three independent experiments. (B) Recombination percentage of episomal fluorescent coding and signal joining substrates comparing XLF-deficient, XLF/ATM doubly deficient, and XLF −/− PAXX −/− 293 cells transiently expressing Rag1, Rag2, and wild-type and mutant forms of XLF as indicated. Error bars indicate SEM from at least 3 independent experiments. Note that XLF L115A shows a significantly lower coding end joining rate in XLF-deficient ( P = 0.0001) and XLF −/− ATM −/− ( P = 0.0022) 293 cells, unlike XLF −/− PAXX −/− cells. Similarly, XLF L115A performs significantly reduced signal joining in XLF-deficient ( P = 0.0210) and XLF −/− ATM −/− ( P = 0.0022) cells, unlike XLF −/− PAXX −/− 293 cells. (C) Zeocin sensitivity of the XLF −/− ATM −/− 293 cell strain stably expressing equivalent levels of wt or mutant XLF. Data from three clones expressing L115A are presented. Error bars indicate SEM from three independent experiments. (D) Immunoblot analysis depicting XLF expression levels in XLF −/− ATM −/− cells stably transfected with wt or mutant XLF or vector control. (E) Immunoblot showing levels of DNA-PKcs autophosphorylation at S2056 with and without zeocin (Zeo) treatment of XLF/ATM doubly deficient cells stably expressing wt and mutant forms of XLF.

    Article Snippet: For the generation of cell strains stably expressing wild-type and mutant constructs of XLF in 293 cells, 5 μg of plasmid DNA was transfected with polyethyleneimine (PEI; 1 μg/ml; Polysciences) at 2 μl/1 μg DNA.

    Techniques: Non-Homologous End Joining, Expressing, Mutagenesis, Stable Transfection, Clone Assay, Transfection, Plasmid Preparation

    XLF L115A does not interact with XRCC4; thus, it does not bridge DNA in vitro but is fully sufficient to stimulate XRCC4/Lig4. (A, left) Schematic of the DNA bridging assay. (Right) Agarose gel showing recovery of DNA fragments bound to streptavidin beads by ethidium bromide staining. Molecular size markers are indicated (kilobases). (B to D) Ethidium bromide staining of agarose gels showing ligation products obtained from in vitro ligation reactions as described in Materials and Methods. Molecular size markers are indicated (kilobases). (B) T4 DNA ligase is utilized. (C) XRCC4/Lig4 complexes (0.4 μM) are utilized. Four different concentrations of XLF were utilized: 0.25 μM, 0.5 μM, 1 μM, and 2 μM. (D) XRCC4/Lig4 complexes (0.2 μM) are utilized, with wild-type or mutant XRCC4 as indicated and with wild-type or mutant XLF (0.5 μM). (E, top) Immunoblot analyses of lysates from 293 cells transiently transfected with His-tagged wt and mutant forms of XLF probed with antibodies to XRCC4, XLF, or Lig4. (Bottom) Immunoblot analyses of pulldown fractions recovered from Ni-NTA–agarose beads after 3 h of incubation of cell lysates with beads and subsequent washing. The immunoblot was probed with antibodies to XRCC4, XLF, or Lig4.

    Journal: Molecular and Cellular Biology

    Article Title: XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation

    doi: 10.1128/MCB.01503-14

    Figure Lengend Snippet: XLF L115A does not interact with XRCC4; thus, it does not bridge DNA in vitro but is fully sufficient to stimulate XRCC4/Lig4. (A, left) Schematic of the DNA bridging assay. (Right) Agarose gel showing recovery of DNA fragments bound to streptavidin beads by ethidium bromide staining. Molecular size markers are indicated (kilobases). (B to D) Ethidium bromide staining of agarose gels showing ligation products obtained from in vitro ligation reactions as described in Materials and Methods. Molecular size markers are indicated (kilobases). (B) T4 DNA ligase is utilized. (C) XRCC4/Lig4 complexes (0.4 μM) are utilized. Four different concentrations of XLF were utilized: 0.25 μM, 0.5 μM, 1 μM, and 2 μM. (D) XRCC4/Lig4 complexes (0.2 μM) are utilized, with wild-type or mutant XRCC4 as indicated and with wild-type or mutant XLF (0.5 μM). (E, top) Immunoblot analyses of lysates from 293 cells transiently transfected with His-tagged wt and mutant forms of XLF probed with antibodies to XRCC4, XLF, or Lig4. (Bottom) Immunoblot analyses of pulldown fractions recovered from Ni-NTA–agarose beads after 3 h of incubation of cell lysates with beads and subsequent washing. The immunoblot was probed with antibodies to XRCC4, XLF, or Lig4.

    Article Snippet: For the generation of cell strains stably expressing wild-type and mutant constructs of XLF in 293 cells, 5 μg of plasmid DNA was transfected with polyethyleneimine (PEI; 1 μg/ml; Polysciences) at 2 μl/1 μg DNA.

    Techniques: In Vitro, Agarose Gel Electrophoresis, Staining, Ligation, Mutagenesis, Transfection, Incubation

    XRCC4/XLF interaction is required for robust DNA-PK autophosphorylation. Immunoblot analysis showing levels of DNA-PKcs autophosphorylation at S2056, total DNA-PKcs, XLF expression, and γH2AX phosphorylation with and without zeocin treatment of 293 cells (left) and XLF-deficient HCT116 cells (right) stably expressing wild-type and mutant forms of XLF.

    Journal: Molecular and Cellular Biology

    Article Title: XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation

    doi: 10.1128/MCB.01503-14

    Figure Lengend Snippet: XRCC4/XLF interaction is required for robust DNA-PK autophosphorylation. Immunoblot analysis showing levels of DNA-PKcs autophosphorylation at S2056, total DNA-PKcs, XLF expression, and γH2AX phosphorylation with and without zeocin treatment of 293 cells (left) and XLF-deficient HCT116 cells (right) stably expressing wild-type and mutant forms of XLF.

    Article Snippet: For the generation of cell strains stably expressing wild-type and mutant constructs of XLF in 293 cells, 5 μg of plasmid DNA was transfected with polyethyleneimine (PEI; 1 μg/ml; Polysciences) at 2 μl/1 μg DNA.

    Techniques: Expressing, Stable Transfection, Mutagenesis

    Characterization of GFP-CLIC5 fusion protein. (A–C) LLC-PK1-CL4 epithelial cell transfected with GFP-CLIC5 ( A ) and counterstained with phalloidin ( B ). Merged images ( C ) with boxed area to indicate region shown in insets. GFP-CLIC5 concentrates in microvilli-like surface structures rich in F-actin. Scale bar: 20 µm. ( D ) Western blot of LLC-PK1-CL4 extracts probed with whole antiserum (B132) against CLIC5. Blot contains two-fold serial dilutions (lanes 8, 4, 2, 1) of extracts from untransfected (untransf), mock-transfected cells treated with transfection reagent with no DNA and GFP-CLIC5 transfected cultures. Bacterial lysate containing 50 ng untagged human CLIC5 (R) was loaded on the same gel as a positive control. A band migrating between ~55 and 60 kDa and corresponding to the expected size of the fusion protein is detected only in the GFP-CLIC5 culture; a faster migrating background band (*) is seen in all cultures. Note that no endogenous CLIC5 (~32 kDa) was detected. ( E ) Western blot stained with Coomassie Blue as a loading control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

    Journal: Cytoskeleton (Hoboken, N.J.)

    Article Title: CLIC5 Stabilizes Membrane-Actin Filament Linkages at the Base of Hair Cell Stereocilia in a Molecular Complex with Radixin, Taperin, and Myosin VI

    doi: 10.1002/cm.21159

    Figure Lengend Snippet: Characterization of GFP-CLIC5 fusion protein. (A–C) LLC-PK1-CL4 epithelial cell transfected with GFP-CLIC5 ( A ) and counterstained with phalloidin ( B ). Merged images ( C ) with boxed area to indicate region shown in insets. GFP-CLIC5 concentrates in microvilli-like surface structures rich in F-actin. Scale bar: 20 µm. ( D ) Western blot of LLC-PK1-CL4 extracts probed with whole antiserum (B132) against CLIC5. Blot contains two-fold serial dilutions (lanes 8, 4, 2, 1) of extracts from untransfected (untransf), mock-transfected cells treated with transfection reagent with no DNA and GFP-CLIC5 transfected cultures. Bacterial lysate containing 50 ng untagged human CLIC5 (R) was loaded on the same gel as a positive control. A band migrating between ~55 and 60 kDa and corresponding to the expected size of the fusion protein is detected only in the GFP-CLIC5 culture; a faster migrating background band (*) is seen in all cultures. Note that no endogenous CLIC5 (~32 kDa) was detected. ( E ) Western blot stained with Coomassie Blue as a loading control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

    Article Snippet: Cells grown on plastic culture dishes or glass coverslips were transfected with plasmid DNA using polyethylenimine (Polysciences, Inc. Warrington, PA) as previously described [ ].

    Techniques: Transfection, Western Blot, Positive Control, Staining

    Hetero- and homomerization of CXCR3 and CXCR4. HEK293T cells were transiently cotransfected with a constant amount (150 ng/10 6 cells) of CXCR4-Rluc (A and B) or CXCR3-Rluc (C and D) DNA and increasing amounts of CXCR3-EYFP (B and D) or CXCR4-EYFP (A and

    Journal: British Journal of Pharmacology

    Article Title: Identification and profiling of CXCR3-CXCR4 chemokine receptor heteromer complexes

    doi: 10.1111/bph.12064

    Figure Lengend Snippet: Hetero- and homomerization of CXCR3 and CXCR4. HEK293T cells were transiently cotransfected with a constant amount (150 ng/10 6 cells) of CXCR4-Rluc (A and B) or CXCR3-Rluc (C and D) DNA and increasing amounts of CXCR3-EYFP (B and D) or CXCR4-EYFP (A and

    Article Snippet: For saturation BRET, time-resolved FRET (trFRET), IP and radioligand-binding studies, HEK293T cells were transfected with indicated amounts of plasmid DNA using 25-kDa linear polyethylenimine (Polysciences, Eppelheim, Germany) as described previously (Verzijl et al ., ).

    Techniques: