pegfp c1  (TaKaRa)


Bioz Verified Symbol TaKaRa is a verified supplier
Bioz Manufacturer Symbol TaKaRa manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    Name:
    Clontech PCR Select Differential Screening Blocking Solution
    Description:

    Catalog Number:
    637402
    Price:
    None
    Category:
    Molecular Biology
    Size:
    1 mL
    Buy from Supplier


    Structured Review

    TaKaRa pegfp c1
    NSAP1 augments HCV IRES-dependent translation. (A) Schematic diagrams of dicistronic reporter plasmids used for monitoring efficiency of cap- and IRES-dependent translation in vivo. The vectors contained the cytomegalovirus (CMV) immediate early enhancer-promoter (CMV pro.) to direct transcription in vivo. The transcription start site in the CMV promoter is indicated by open arrows. (B) (i) Translation activities of IRES-containing dicistronic mRNAs in 293T cells. Dicistronic reporter constructs (0.5 μg each of pRH402F [lanes 1 to 4], pRPF [lanes 5 to 8], pREF [lanes 9 to 12], and pRM531F [lanes 13 to 16]) and variable amounts of effecter <t>pEGFP-c1/NSAP1</t> (2.5 μg [lanes 2, 6, 10, and 14], 5 μg [lanes 3, 7, 11, and 15], and 10 μg [lanes 4, 8, 12, and 16]) and negative control effecter pEGFP-c1 (10 μg [lanes 1, 5, 9, and 13], 7.5 μg [lanes 2, 6, 10, and 14], and 5 μg [lanes 3, 7, 11, and 15]) were cotransfected with the control plasmid pCMV · SPORT-βgal (Invitrogen) to monitor the effect of NSAP1 on IRES activities. Forty-eight hours after transfection by electroporation, the cells were harvested and their luciferase activities were measured. For IRES-dependent translation, firefly luciferase activity, which was directed by pRH402F, was arbitrarily set to 1. For cap-dependent translation, Renilla luciferase activity, which was directed by pRH402F, was arbitrarily set to 1. The transfection efficiency was also determined for each luciferase value relative to that of the β-galactosidase activity of the transfection control. Error bars represent standard deviation values. (ii) Expression profiles of GFP-NSAP1. Immunoblot analysis was performed with antibodies against GFP, NSAP1, and actin.

    https://www.bioz.com/result/pegfp c1/product/TaKaRa
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pegfp c1 - by Bioz Stars, 2021-06
    97/100 stars

    Images

    1) Product Images from "A Cellular RNA-Binding Protein Enhances Internal Ribosomal Entry Site-Dependent Translation through an Interaction Downstream of the Hepatitis C Virus Polyprotein Initiation Codon"

    Article Title: A Cellular RNA-Binding Protein Enhances Internal Ribosomal Entry Site-Dependent Translation through an Interaction Downstream of the Hepatitis C Virus Polyprotein Initiation Codon

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.18.7878-7890.2004

    NSAP1 augments HCV IRES-dependent translation. (A) Schematic diagrams of dicistronic reporter plasmids used for monitoring efficiency of cap- and IRES-dependent translation in vivo. The vectors contained the cytomegalovirus (CMV) immediate early enhancer-promoter (CMV pro.) to direct transcription in vivo. The transcription start site in the CMV promoter is indicated by open arrows. (B) (i) Translation activities of IRES-containing dicistronic mRNAs in 293T cells. Dicistronic reporter constructs (0.5 μg each of pRH402F [lanes 1 to 4], pRPF [lanes 5 to 8], pREF [lanes 9 to 12], and pRM531F [lanes 13 to 16]) and variable amounts of effecter pEGFP-c1/NSAP1 (2.5 μg [lanes 2, 6, 10, and 14], 5 μg [lanes 3, 7, 11, and 15], and 10 μg [lanes 4, 8, 12, and 16]) and negative control effecter pEGFP-c1 (10 μg [lanes 1, 5, 9, and 13], 7.5 μg [lanes 2, 6, 10, and 14], and 5 μg [lanes 3, 7, 11, and 15]) were cotransfected with the control plasmid pCMV · SPORT-βgal (Invitrogen) to monitor the effect of NSAP1 on IRES activities. Forty-eight hours after transfection by electroporation, the cells were harvested and their luciferase activities were measured. For IRES-dependent translation, firefly luciferase activity, which was directed by pRH402F, was arbitrarily set to 1. For cap-dependent translation, Renilla luciferase activity, which was directed by pRH402F, was arbitrarily set to 1. The transfection efficiency was also determined for each luciferase value relative to that of the β-galactosidase activity of the transfection control. Error bars represent standard deviation values. (ii) Expression profiles of GFP-NSAP1. Immunoblot analysis was performed with antibodies against GFP, NSAP1, and actin.
    Figure Legend Snippet: NSAP1 augments HCV IRES-dependent translation. (A) Schematic diagrams of dicistronic reporter plasmids used for monitoring efficiency of cap- and IRES-dependent translation in vivo. The vectors contained the cytomegalovirus (CMV) immediate early enhancer-promoter (CMV pro.) to direct transcription in vivo. The transcription start site in the CMV promoter is indicated by open arrows. (B) (i) Translation activities of IRES-containing dicistronic mRNAs in 293T cells. Dicistronic reporter constructs (0.5 μg each of pRH402F [lanes 1 to 4], pRPF [lanes 5 to 8], pREF [lanes 9 to 12], and pRM531F [lanes 13 to 16]) and variable amounts of effecter pEGFP-c1/NSAP1 (2.5 μg [lanes 2, 6, 10, and 14], 5 μg [lanes 3, 7, 11, and 15], and 10 μg [lanes 4, 8, 12, and 16]) and negative control effecter pEGFP-c1 (10 μg [lanes 1, 5, 9, and 13], 7.5 μg [lanes 2, 6, 10, and 14], and 5 μg [lanes 3, 7, 11, and 15]) were cotransfected with the control plasmid pCMV · SPORT-βgal (Invitrogen) to monitor the effect of NSAP1 on IRES activities. Forty-eight hours after transfection by electroporation, the cells were harvested and their luciferase activities were measured. For IRES-dependent translation, firefly luciferase activity, which was directed by pRH402F, was arbitrarily set to 1. For cap-dependent translation, Renilla luciferase activity, which was directed by pRH402F, was arbitrarily set to 1. The transfection efficiency was also determined for each luciferase value relative to that of the β-galactosidase activity of the transfection control. Error bars represent standard deviation values. (ii) Expression profiles of GFP-NSAP1. Immunoblot analysis was performed with antibodies against GFP, NSAP1, and actin.

    Techniques Used: In Vivo, Construct, Negative Control, Plasmid Preparation, Transfection, Electroporation, Luciferase, Activity Assay, Standard Deviation, Expressing

    2) Product Images from "FRL, a Novel Formin-Related Protein, Binds to Rac and Regulates Cell Motility and Survival of Macrophages"

    Article Title: FRL, a Novel Formin-Related Protein, Binds to Rac and Regulates Cell Motility and Survival of Macrophages

    Journal: Molecular and Cellular Biology

    doi:

    Association of FRL and truncated FRL proteins with Rac1 in vitro and in vivo. (A) Precipitation of FRL by the GTPγS- or GDP-bound form of Rac1. P388D1 cell lysates were incubated with GST-RhoA, GST-Rac1, GST-Cdc42Hs, or GST, each of which was preloaded with either GTPγs or GDP. GST fusion proteins were precipitated by glutathione-Sepharose 4B beads, and the pellets were analyzed by immunoblotting with an anti-FRL antibody. (B) In vivo association of FRL with Rac1. P388D1 cell lysates were immobilized on protein A-agarose beads without (lane 1) or with (lane 2) anti-Rac1. The precipitates were analyzed by Western blotting with an anti-FRL antibody and an anti-Rac1 antibody. (C) Structure of truncated FRLs. FH3-FRL contains only the FH3 domain, FH1+FH3-FRL contains the FH1 and FH3 domains, and FH2-FRL contains only the FH2 domain. N1 consists of the N-terminal 80 amino acids, and N2 consists of the N-terminal 216 amino acids. (D) FRL associates with Rac1 at its N-terminal region containing the FH3 domain. COS7 cells transfected with each expression vector (FH3-pEGFP-C1, FH1+FH3-pEGFP-C1, FH2-pEGFP-C1, N1-pEGFP-C1, or N2-pEGFP-C1) were lysed, incubated with GST-Rac1, and precipitated by glutathione-Sepharose 4B. The pellets were analyzed by immunoblotting with an anti-GFP antibody.
    Figure Legend Snippet: Association of FRL and truncated FRL proteins with Rac1 in vitro and in vivo. (A) Precipitation of FRL by the GTPγS- or GDP-bound form of Rac1. P388D1 cell lysates were incubated with GST-RhoA, GST-Rac1, GST-Cdc42Hs, or GST, each of which was preloaded with either GTPγs or GDP. GST fusion proteins were precipitated by glutathione-Sepharose 4B beads, and the pellets were analyzed by immunoblotting with an anti-FRL antibody. (B) In vivo association of FRL with Rac1. P388D1 cell lysates were immobilized on protein A-agarose beads without (lane 1) or with (lane 2) anti-Rac1. The precipitates were analyzed by Western blotting with an anti-FRL antibody and an anti-Rac1 antibody. (C) Structure of truncated FRLs. FH3-FRL contains only the FH3 domain, FH1+FH3-FRL contains the FH1 and FH3 domains, and FH2-FRL contains only the FH2 domain. N1 consists of the N-terminal 80 amino acids, and N2 consists of the N-terminal 216 amino acids. (D) FRL associates with Rac1 at its N-terminal region containing the FH3 domain. COS7 cells transfected with each expression vector (FH3-pEGFP-C1, FH1+FH3-pEGFP-C1, FH2-pEGFP-C1, N1-pEGFP-C1, or N2-pEGFP-C1) were lysed, incubated with GST-Rac1, and precipitated by glutathione-Sepharose 4B. The pellets were analyzed by immunoblotting with an anti-GFP antibody.

    Techniques Used: In Vitro, In Vivo, Incubation, Western Blot, Transfection, Expressing, Plasmid Preparation

    FRL associates with profilin. (A) Interaction of FRL and profilin in vitro. P388D1 cell lysates were incubated with GST-profilin I or with GST-profilin II and then precipitated by glutathione-Sepharose 4B beads, and the pellets were analyzed by immunoblotting with anti-FRL, anti-GST, and anti-actin antibodies. (B) In vivo association of FRL with profilin. P388D1 cell lysates were immobilized on protein A-agarose beads without (lane 1) or with (lane 2) an anti-FRL antibody. The precipitates were analyzed by Western blotting with anti-FRL and anti-profilin antibodies. (C) The FH1 domain of FRL binds to profilin. COS7 cells transfected with each expression vector (FH3-pEGFP-C1, FH1+FH3-pEGFP-C1, and FH2-pEGFP-C1) were lysed, incubated with GST-profilin I, and precipitated by glutathione-Sepharose 4B beads. The pellets were analyzed by immunoblotting with anti-GFP and anti-actin antibodies.
    Figure Legend Snippet: FRL associates with profilin. (A) Interaction of FRL and profilin in vitro. P388D1 cell lysates were incubated with GST-profilin I or with GST-profilin II and then precipitated by glutathione-Sepharose 4B beads, and the pellets were analyzed by immunoblotting with anti-FRL, anti-GST, and anti-actin antibodies. (B) In vivo association of FRL with profilin. P388D1 cell lysates were immobilized on protein A-agarose beads without (lane 1) or with (lane 2) an anti-FRL antibody. The precipitates were analyzed by Western blotting with anti-FRL and anti-profilin antibodies. (C) The FH1 domain of FRL binds to profilin. COS7 cells transfected with each expression vector (FH3-pEGFP-C1, FH1+FH3-pEGFP-C1, and FH2-pEGFP-C1) were lysed, incubated with GST-profilin I, and precipitated by glutathione-Sepharose 4B beads. The pellets were analyzed by immunoblotting with anti-GFP and anti-actin antibodies.

    Techniques Used: In Vitro, Incubation, In Vivo, Western Blot, Transfection, Expressing, Plasmid Preparation

    Overexpression of FH1+FH3-FRL or FULL-FRL protein restored the cell adhesion, migration, and proliferation abilities of doxycycline-treated FH3/P388D1 cells. FH3/P388D1 cells were transfected with FH1+FH3-pEGFP-C1 (FH1+FH3), FULL-pEGFP-C1 (FULL), and pEGFP-C1 vector only (control), and then cultured with doxycycline for 24 h. (A) The cells expressing FH1+FH3-FRL or FULL-FRL together with FH3-FRL truncated protein spread and formed ruffles upon stimulation with SDF-1. In the panels on the left, cells were stained with rhodamine-conjugated phalloidin for actin staining. Right panels show GFP-positive cells. Note that the cell without expression of transfected FRL-GFP fusion protein does not show spreading or the formation of membrane ruffles. (B) Adhesion assay. Expression of FH1+FH3 or FULL-FRL also rescued the failure of FH3/P388D1 cells to adhere to fibronectin. (C) Migration of FH3/P388D1 against SDF-1 after expression of FH1+FH3-FRL or FULL-FRL protein together with FH3-FRL truncated protein. (D) [ 3 H]thymidine incorporation of FULL-FRL- or FH1+FH3-FRL-transfected FH3/P388D1 cells at 72 h after the addition of doxycycline (1.0 μg/ml). Proliferation of FH3/P388D1 cells was restored by expression of FH1+FH3-FRL or FULL-FRL protein, even in the presence of the truncated FH3-FRL induced by doxycycline.
    Figure Legend Snippet: Overexpression of FH1+FH3-FRL or FULL-FRL protein restored the cell adhesion, migration, and proliferation abilities of doxycycline-treated FH3/P388D1 cells. FH3/P388D1 cells were transfected with FH1+FH3-pEGFP-C1 (FH1+FH3), FULL-pEGFP-C1 (FULL), and pEGFP-C1 vector only (control), and then cultured with doxycycline for 24 h. (A) The cells expressing FH1+FH3-FRL or FULL-FRL together with FH3-FRL truncated protein spread and formed ruffles upon stimulation with SDF-1. In the panels on the left, cells were stained with rhodamine-conjugated phalloidin for actin staining. Right panels show GFP-positive cells. Note that the cell without expression of transfected FRL-GFP fusion protein does not show spreading or the formation of membrane ruffles. (B) Adhesion assay. Expression of FH1+FH3 or FULL-FRL also rescued the failure of FH3/P388D1 cells to adhere to fibronectin. (C) Migration of FH3/P388D1 against SDF-1 after expression of FH1+FH3-FRL or FULL-FRL protein together with FH3-FRL truncated protein. (D) [ 3 H]thymidine incorporation of FULL-FRL- or FH1+FH3-FRL-transfected FH3/P388D1 cells at 72 h after the addition of doxycycline (1.0 μg/ml). Proliferation of FH3/P388D1 cells was restored by expression of FH1+FH3-FRL or FULL-FRL protein, even in the presence of the truncated FH3-FRL induced by doxycycline.

    Techniques Used: Over Expression, Migration, Transfection, Plasmid Preparation, Cell Culture, Expressing, Staining, Cell Adhesion Assay

    3) Product Images from "Intracellular Distributing and Interferon-? Secretion of Human Interleukin-18 in BxPC-3 Cells"

    Article Title: Intracellular Distributing and Interferon-? Secretion of Human Interleukin-18 in BxPC-3 Cells

    Journal: International Journal of Medical Sciences

    doi: 10.7150/ijms.6875

    RT-PCR products of five recombinant plasmids. M: 100-6000 bp DNA Marker. (A) Lane 1-20 showed the RT-PCR results of BxPC-3 cells tranfected with five recombinant plasmids (Mu0, Mu1, Mu2, Mu3,Mu4) at 12 h, 24 h, 36 h, 48 h respectively, using the primer of β-actin. (B) Lane 1-20 showed the RT-PCR results of BxPC-3 cells tranfected with five recombinant plasmids (Mu0, Mu1, Mu2, Mu3, Mu4) at 12 h, 24 h, 36 h, 48 h respectively, using the primer of hIL-18 full length. Lane 21 is negative control. (C) Lane1-20 showed the RT-PCR results of BxPC-3 cells tranfected with five recombinant plasmids (Mu0, Mu1, Mu2, Mu3, Mu4) at 12 h, 24 h, 36 h, 48 h respectively, using the original primers. (D) Control. Lane 1-4 and lane 5-8 showed RT-PCR results of BxPC-3 cells tranfected with pEGFP-C1 and untreated BxPC-3 cells cultivated at 12 h, 24 h, 36 h, 48 h respectively, using the primer of hIL-18 full length. Lane 9-12 and lane 13-16 showed RT-PCR results of BxPC-3 cells with the same treatment (according to Lane 1-4 and 5-8) using the primer of β-actin.
    Figure Legend Snippet: RT-PCR products of five recombinant plasmids. M: 100-6000 bp DNA Marker. (A) Lane 1-20 showed the RT-PCR results of BxPC-3 cells tranfected with five recombinant plasmids (Mu0, Mu1, Mu2, Mu3,Mu4) at 12 h, 24 h, 36 h, 48 h respectively, using the primer of β-actin. (B) Lane 1-20 showed the RT-PCR results of BxPC-3 cells tranfected with five recombinant plasmids (Mu0, Mu1, Mu2, Mu3, Mu4) at 12 h, 24 h, 36 h, 48 h respectively, using the primer of hIL-18 full length. Lane 21 is negative control. (C) Lane1-20 showed the RT-PCR results of BxPC-3 cells tranfected with five recombinant plasmids (Mu0, Mu1, Mu2, Mu3, Mu4) at 12 h, 24 h, 36 h, 48 h respectively, using the original primers. (D) Control. Lane 1-4 and lane 5-8 showed RT-PCR results of BxPC-3 cells tranfected with pEGFP-C1 and untreated BxPC-3 cells cultivated at 12 h, 24 h, 36 h, 48 h respectively, using the primer of hIL-18 full length. Lane 9-12 and lane 13-16 showed RT-PCR results of BxPC-3 cells with the same treatment (according to Lane 1-4 and 5-8) using the primer of β-actin.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Recombinant, Marker, Negative Control

    Localization of enhanced green fluorescent protein (EGFP)-tagged hIL-18 recombinant plasmids inBxPC-3 cells. Confocal micrographs of transfected BxPC-3 cells expressing EGFP-tagged hIL-18 recombinant plasmid at 12 h post-transfection (A) 24 h post-transfection, (B) 36 h post-transfection, (C) 48 h post-transfection, (D) and composite photo (E) respectively . Images were taken in the plane where maximum fluorescence appeared. Transfected cells presented uniform fluorescence throughout the cytoplasm and the nucleus 12 h post-transfection, and the fluorescence intensity of transfected cells with pEGFP-C1 are durable(A-D), fluorescence intensity of other cells are decreasing. But from 24 h and 36 h post-transfection, the fluorescence of the hIL-18 Mu1 and Mu2 appeared targeted to the membranous region of the BxPC-3 cells. Magnification, 200×.
    Figure Legend Snippet: Localization of enhanced green fluorescent protein (EGFP)-tagged hIL-18 recombinant plasmids inBxPC-3 cells. Confocal micrographs of transfected BxPC-3 cells expressing EGFP-tagged hIL-18 recombinant plasmid at 12 h post-transfection (A) 24 h post-transfection, (B) 36 h post-transfection, (C) 48 h post-transfection, (D) and composite photo (E) respectively . Images were taken in the plane where maximum fluorescence appeared. Transfected cells presented uniform fluorescence throughout the cytoplasm and the nucleus 12 h post-transfection, and the fluorescence intensity of transfected cells with pEGFP-C1 are durable(A-D), fluorescence intensity of other cells are decreasing. But from 24 h and 36 h post-transfection, the fluorescence of the hIL-18 Mu1 and Mu2 appeared targeted to the membranous region of the BxPC-3 cells. Magnification, 200×.

    Techniques Used: Recombinant, Transfection, Expressing, Plasmid Preparation, Fluorescence

    4) Product Images from "Human VPS13A is associated with multiple organelles and influences mitochondrial morphology and lipid droplet motility"

    Article Title: Human VPS13A is associated with multiple organelles and influences mitochondrial morphology and lipid droplet motility

    Journal: eLife

    doi: 10.7554/eLife.43561

    VPS13A interacts with VAP-A in human cells. ( A ) GFP-VPS13A constructs presented in Figure 2D–E were overexpressed in HEK293T cells for 24 hr. Cell lysates were processed for immunoblot analysis using an antibody against GFP. peGFP-C1 (GFP) expressing cells were used as a control. The stain free gel is shown as a loading control. B,C GFP-VPS13 (2003–2606) and GFP-VPS13A (2615–3174) constructs were expressed in either HEK293T cells (B/B’) or U2OS cells (C/C’). Note the differences in mitochondria morphology in both cell types. ( D ) U2OS cells expressing GFP-VPS13A (2615–3174) for 24 hr were stained with Mitotracker Red. D’ shows a higher magnification of the insert in D. Cells were co-transfected with mCherrySec61 (B/C) and BFP-Sec61 (D/D’) (not shown). Scale bars = 25 μm ( B–C’ ) and 10 μm ( D–D’ ).
    Figure Legend Snippet: VPS13A interacts with VAP-A in human cells. ( A ) GFP-VPS13A constructs presented in Figure 2D–E were overexpressed in HEK293T cells for 24 hr. Cell lysates were processed for immunoblot analysis using an antibody against GFP. peGFP-C1 (GFP) expressing cells were used as a control. The stain free gel is shown as a loading control. B,C GFP-VPS13 (2003–2606) and GFP-VPS13A (2615–3174) constructs were expressed in either HEK293T cells (B/B’) or U2OS cells (C/C’). Note the differences in mitochondria morphology in both cell types. ( D ) U2OS cells expressing GFP-VPS13A (2615–3174) for 24 hr were stained with Mitotracker Red. D’ shows a higher magnification of the insert in D. Cells were co-transfected with mCherrySec61 (B/C) and BFP-Sec61 (D/D’) (not shown). Scale bars = 25 μm ( B–C’ ) and 10 μm ( D–D’ ).

    Techniques Used: Construct, Expressing, Staining, Transfection

    VPS13A colocalizes with mitochondria but not with the endocytic compartment. ( A ) 48 hours after transfection with either VPS13A-Myc or VPS13A-GFP, HEK293T cells were processed for immunoblotting using antibodies against VPS13A and a -Tubulin. peGFP-C1 transfected or non-transfected (NT) cells were used as controls. Note the enrichment of VPS13A in both VPS13A-Myc or VPS13A-GFP lanes. ( B ) Quantification of protein bands detected with anti-VPS13A antibody in A. The ratio of VPS13A to a -tubulin was normalized to NT cells. Error bars, mean ± s.e.m (n=3), two-tailed unpaired Student’s t-test was used (*P ≤ 0.05, **P≤0.01). C-F HEK293T cells were co-transfected with VPS13A-Myc and with GFP-Rab5 Q79L ( C ), GFP-Rab7 Q67L ( D ), LAMP1-GFP ( E ) or mCherry FYCO1 ( F ). Cells were stained with anti-myc (C-E, red; F, green) and DAPI (blue). Bottom panels (C’-F’ show a magnification of the inset in top panels. Scale bars = 10 µm ( C–F’ ).
    Figure Legend Snippet: VPS13A colocalizes with mitochondria but not with the endocytic compartment. ( A ) 48 hours after transfection with either VPS13A-Myc or VPS13A-GFP, HEK293T cells were processed for immunoblotting using antibodies against VPS13A and a -Tubulin. peGFP-C1 transfected or non-transfected (NT) cells were used as controls. Note the enrichment of VPS13A in both VPS13A-Myc or VPS13A-GFP lanes. ( B ) Quantification of protein bands detected with anti-VPS13A antibody in A. The ratio of VPS13A to a -tubulin was normalized to NT cells. Error bars, mean ± s.e.m (n=3), two-tailed unpaired Student’s t-test was used (*P ≤ 0.05, **P≤0.01). C-F HEK293T cells were co-transfected with VPS13A-Myc and with GFP-Rab5 Q79L ( C ), GFP-Rab7 Q67L ( D ), LAMP1-GFP ( E ) or mCherry FYCO1 ( F ). Cells were stained with anti-myc (C-E, red; F, green) and DAPI (blue). Bottom panels (C’-F’ show a magnification of the inset in top panels. Scale bars = 10 µm ( C–F’ ).

    Techniques Used: Transfection, Two Tailed Test, Staining

    VPS13A interacts with VAP-A. ( A ) GST-fusion proteins of VPS13A fragments expressed in E.Coli were enriched on Sepharose beads and incubated with equal amounts of HeLa cell lysate. GST alone was used as a control. Samples were immunoblotted against VAP-A, GST and N-terminal VPS13A (H-102). ( B ) Full length VPS13A-GFP ( B ) or VPS13A ∆FFAT -GFP ( B’ ) were expressed in HEK293T cells and mitochondria were marked using an antibody against TOMM20. The yellow signal in the overlay represents sites of close association between mitochondria and the VPS13A-GFP. Cells were co-transfected with BFP-Sec61 (not shown). ( C ) HEK293T cells were transfected with VPS13A-GFP or VPS13A ∆FFAT -GFP and stained for TOMM20. The fraction of the GFP signal overlapping the TOMM20 signal was quantified with ImageJ using the JACoP plugin. ( D ) HEK293T cells were transfected with VPS13A-GFP or VPS13A ∆FFAT -GFP and mCherry-VAP-A. The fraction of the GFP signal overlapping the mCherry signal was quantified with ImageJ using the JACoP plugin. Error bars ( C, D ), mean ±s.e.m (n = 3), two-tailed unpaired Student’s t-test was used (**p≤0.01). ( E ) Full length VPS13A-GFP, VPS13A ∆FFAT -GFP or peGFP-C1 (as a control) were expressed in HEK293T cells and immunoprecipitated using a GFP-trap assay. Samples from Figure 4F were co-analysed for the presence of VAP-B. Scale bar = 10 µm (B/B’).
    Figure Legend Snippet: VPS13A interacts with VAP-A. ( A ) GST-fusion proteins of VPS13A fragments expressed in E.Coli were enriched on Sepharose beads and incubated with equal amounts of HeLa cell lysate. GST alone was used as a control. Samples were immunoblotted against VAP-A, GST and N-terminal VPS13A (H-102). ( B ) Full length VPS13A-GFP ( B ) or VPS13A ∆FFAT -GFP ( B’ ) were expressed in HEK293T cells and mitochondria were marked using an antibody against TOMM20. The yellow signal in the overlay represents sites of close association between mitochondria and the VPS13A-GFP. Cells were co-transfected with BFP-Sec61 (not shown). ( C ) HEK293T cells were transfected with VPS13A-GFP or VPS13A ∆FFAT -GFP and stained for TOMM20. The fraction of the GFP signal overlapping the TOMM20 signal was quantified with ImageJ using the JACoP plugin. ( D ) HEK293T cells were transfected with VPS13A-GFP or VPS13A ∆FFAT -GFP and mCherry-VAP-A. The fraction of the GFP signal overlapping the mCherry signal was quantified with ImageJ using the JACoP plugin. Error bars ( C, D ), mean ±s.e.m (n = 3), two-tailed unpaired Student’s t-test was used (**p≤0.01). ( E ) Full length VPS13A-GFP, VPS13A ∆FFAT -GFP or peGFP-C1 (as a control) were expressed in HEK293T cells and immunoprecipitated using a GFP-trap assay. Samples from Figure 4F were co-analysed for the presence of VAP-B. Scale bar = 10 µm (B/B’).

    Techniques Used: Incubation, Transfection, Staining, Two Tailed Test, Immunoprecipitation, TRAP Assay

    5) Product Images from "Zipcode Binding Protein 1 Regulates the Development of Dendritic Arbors in Hippocampal Neurons"

    Article Title: Zipcode Binding Protein 1 Regulates the Development of Dendritic Arbors in Hippocampal Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2387-10.2011

    β-Actin overexpression in hippocampal neurons with ZBP1 knockdown is sufficient for partial phenotype rescue. A , Micrographs of hippocampal neurons transfected on DIV7 with pEGFP-C1 together with pSuper vector as a control, ZBP1sh#1, or ZBP1sh#1 together with EGFP-ZBP1*1 or EGFP-β-actin. Expression proceeded for 3 d. Neuronal morphology was visualized by staining for cotransfected β-gal. B–F , Mean number of primary dendrites ( B ), mean number of dendritic tips ( C ), dendritic branching index ( D ), mean total dendritic length ( E ), and Sholl analysis of transfected neurons ( F ). *** p
    Figure Legend Snippet: β-Actin overexpression in hippocampal neurons with ZBP1 knockdown is sufficient for partial phenotype rescue. A , Micrographs of hippocampal neurons transfected on DIV7 with pEGFP-C1 together with pSuper vector as a control, ZBP1sh#1, or ZBP1sh#1 together with EGFP-ZBP1*1 or EGFP-β-actin. Expression proceeded for 3 d. Neuronal morphology was visualized by staining for cotransfected β-gal. B–F , Mean number of primary dendrites ( B ), mean number of dendritic tips ( C ), dendritic branching index ( D ), mean total dendritic length ( E ), and Sholl analysis of transfected neurons ( F ). *** p

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Expressing, Staining

    Characterization of ZBP1 shRNAs. A–C , shRNA and scrambled RNA validation in COS-7 cell line. Cells were transfected with control or shRNA encoding plasmid together with either EGFP-ZBP1 or pEGFP-C1. Additionally, β-gal was cotransfected as a marker of even transfection. Both ZBP1sh#1 and ZBP1sh#2 strongly reduced EGFP-ZBP1 expression, whereas the scrambled version (scZBP1sh) did not. D–F , Verification of specificity of ZBP1shRNAs using qRT-PCR of cDNAs from neurons transfected on DIV0 with nucleofection with pSuper, ZBP1sh#1, ZBP1sh#2, and scrambled RNA. The graphs show the relative quantification of ZBP1 and two other members of the ZBP1 family: IMP2 and IMP3. * p
    Figure Legend Snippet: Characterization of ZBP1 shRNAs. A–C , shRNA and scrambled RNA validation in COS-7 cell line. Cells were transfected with control or shRNA encoding plasmid together with either EGFP-ZBP1 or pEGFP-C1. Additionally, β-gal was cotransfected as a marker of even transfection. Both ZBP1sh#1 and ZBP1sh#2 strongly reduced EGFP-ZBP1 expression, whereas the scrambled version (scZBP1sh) did not. D–F , Verification of specificity of ZBP1shRNAs using qRT-PCR of cDNAs from neurons transfected on DIV0 with nucleofection with pSuper, ZBP1sh#1, ZBP1sh#2, and scrambled RNA. The graphs show the relative quantification of ZBP1 and two other members of the ZBP1 family: IMP2 and IMP3. * p

    Techniques Used: shRNA, Transfection, Plasmid Preparation, Marker, Expressing, Quantitative RT-PCR

    ZBP1 mutants with impaired RNA binding do not affect dendritic branching when overexpressed and are not sufficient for phenotype rescue in neurons with endogenous ZBP1 knockdown. A , Schematic representations of mutants used in this set of experiments. B , Representative micrographs of hippocampal neurons transfected on DIV7 with control vector, EGFP-ZBP1*1, EGFP-ZBP1*1ΔC, or EGFP-ZBP1*1-GXXG. Neuronal morphology was visualized by staining for cotransfected β-gal. Scale bar, 20 μm. C–G , Mean number of primary dendrites ( C ), mean total number of dendritic tips ( D ), dendritic branching index ( E ), total length of dendrites ( F ), and Sholl analysis of neurons overexpressing EGFP, EGFP-ZBP1*1, EGFP-ZBP1*1ΔC, or EGFP-ZBP1*1-GXXG ( G ). H , Representative micrographs of hippocampal neurons transfected on DIV7 with pEGFP-C1 together with empty pSuper vector as a control, ZBP1sh#1, or ZBP1sh#1 together with EGFP-ZBP1*1, EGFP-ZBP1*1ΔC, or EGFP-ZBP1*1-GXXG. Expression proceeded for 3 d. Neuronal morphology was visualized by staining for cotransfected β-gal. Scale bar, 20 μm. I–K , Mean number of primary dendrites ( I ), mean total number of dendritic tips ( J ), and dendritic branching index of transfected neurons ( K ). *** p
    Figure Legend Snippet: ZBP1 mutants with impaired RNA binding do not affect dendritic branching when overexpressed and are not sufficient for phenotype rescue in neurons with endogenous ZBP1 knockdown. A , Schematic representations of mutants used in this set of experiments. B , Representative micrographs of hippocampal neurons transfected on DIV7 with control vector, EGFP-ZBP1*1, EGFP-ZBP1*1ΔC, or EGFP-ZBP1*1-GXXG. Neuronal morphology was visualized by staining for cotransfected β-gal. Scale bar, 20 μm. C–G , Mean number of primary dendrites ( C ), mean total number of dendritic tips ( D ), dendritic branching index ( E ), total length of dendrites ( F ), and Sholl analysis of neurons overexpressing EGFP, EGFP-ZBP1*1, EGFP-ZBP1*1ΔC, or EGFP-ZBP1*1-GXXG ( G ). H , Representative micrographs of hippocampal neurons transfected on DIV7 with pEGFP-C1 together with empty pSuper vector as a control, ZBP1sh#1, or ZBP1sh#1 together with EGFP-ZBP1*1, EGFP-ZBP1*1ΔC, or EGFP-ZBP1*1-GXXG. Expression proceeded for 3 d. Neuronal morphology was visualized by staining for cotransfected β-gal. Scale bar, 20 μm. I–K , Mean number of primary dendrites ( I ), mean total number of dendritic tips ( J ), and dendritic branching index of transfected neurons ( K ). *** p

    Techniques Used: RNA Binding Assay, Transfection, Plasmid Preparation, Staining, Expressing

    6) Product Images from "Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids"

    Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043283

    Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
    Figure Legend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

    Techniques Used: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

    Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.
    Figure Legend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

    Techniques Used: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

    7) Product Images from "Evolutionary insights into T-type Ca2+ channel structure, function, and ion selectivity from the Trichoplax adhaerens homologue"

    Article Title: Evolutionary insights into T-type Ca2+ channel structure, function, and ion selectivity from the Trichoplax adhaerens homologue

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201611683

    In vitro expression of TCa v 3 in HEK-293T cells. (A) Bicistronic EGFP fluorescence images (top) and merged EGFP/transmitted light images (bottom) of HEK-293T cells cotransfected with pTCa v 3-IR-EGFP (vector map on left) and pCDNA-3.1 (+pC) and/or pMT2 constructs bearing the coding sequences of rat Ca v β 1b and Ca v α 2 δ 1 accessory subunits. (B) Similar to A, but cells were transfected with the pEGFP-TCa v 3 construct, encoding a fusion protein of TCa v 3 tagged with N-terminal EGFP. (C) Similar to A, but cells were transfected with the empty EGFP fusion vector pEGFP-C1. Bar, 200 µm. (D) Bar graph depicting normalized mean fluorescence of imaged HEK cells from quadruplicate transfections (±SE). The purple asterisks denote statistically significant means (***, P
    Figure Legend Snippet: In vitro expression of TCa v 3 in HEK-293T cells. (A) Bicistronic EGFP fluorescence images (top) and merged EGFP/transmitted light images (bottom) of HEK-293T cells cotransfected with pTCa v 3-IR-EGFP (vector map on left) and pCDNA-3.1 (+pC) and/or pMT2 constructs bearing the coding sequences of rat Ca v β 1b and Ca v α 2 δ 1 accessory subunits. (B) Similar to A, but cells were transfected with the pEGFP-TCa v 3 construct, encoding a fusion protein of TCa v 3 tagged with N-terminal EGFP. (C) Similar to A, but cells were transfected with the empty EGFP fusion vector pEGFP-C1. Bar, 200 µm. (D) Bar graph depicting normalized mean fluorescence of imaged HEK cells from quadruplicate transfections (±SE). The purple asterisks denote statistically significant means (***, P

    Techniques Used: In Vitro, Expressing, Fluorescence, Plasmid Preparation, Construct, Transfection

    8) Product Images from "Potential of Herpesvirus Saimiri-Based Vectors To Reprogram a Somatic Ewing's Sarcoma Family Tumor Cell Line"

    Article Title: Potential of Herpesvirus Saimiri-Based Vectors To Reprogram a Somatic Ewing's Sarcoma Family Tumor Cell Line

    Journal: Journal of Virology

    doi: 10.1128/JVI.03147-12

    Generation and characterization of HVS-iPSC-BAC recombinant viruses. (A) Schematic representation of the cloning procedure. iPSC genes were PCR amplified, and the resulting products were cloned into pEGFP-c1, replacing the EGFP coding region, to produce pCMV-iPSC constructs, thereby placing iPSC genes under the control of the CMV IE promoter. CMV-iPSC expression cassettes were PCR amplified and cloned into the pShuttle Link 1 vector prior to subcloning into predigested HVS-GFP-BAC at flanking I-PpoI restriction sites. (B) PFGE of HVS-iPSC-BAC recombinants. The presence of the iPSC expression cassettes was confirmed by I-PpoI restriction digestion (white arrows). (C) 293T cells were either transfected with pCMV-iPSC constructs or infected with each HVS-iPSC viral vector expressing Oct4, Lin28, or Nanog at an MOI of 1. After 24 h, the cell lysates were immunoblotted with Oct4-, Lin28-, and Nanog-specific antibodies. Negative controls included pEGFP-c1-transfected cells and HVS-GFP-infected cells. Expression of iPSC transgenes is confirmed by the presence of specific bands at 39, 23, and 34 kDa for Oct4, Lin28, and Nanog, respectively.
    Figure Legend Snippet: Generation and characterization of HVS-iPSC-BAC recombinant viruses. (A) Schematic representation of the cloning procedure. iPSC genes were PCR amplified, and the resulting products were cloned into pEGFP-c1, replacing the EGFP coding region, to produce pCMV-iPSC constructs, thereby placing iPSC genes under the control of the CMV IE promoter. CMV-iPSC expression cassettes were PCR amplified and cloned into the pShuttle Link 1 vector prior to subcloning into predigested HVS-GFP-BAC at flanking I-PpoI restriction sites. (B) PFGE of HVS-iPSC-BAC recombinants. The presence of the iPSC expression cassettes was confirmed by I-PpoI restriction digestion (white arrows). (C) 293T cells were either transfected with pCMV-iPSC constructs or infected with each HVS-iPSC viral vector expressing Oct4, Lin28, or Nanog at an MOI of 1. After 24 h, the cell lysates were immunoblotted with Oct4-, Lin28-, and Nanog-specific antibodies. Negative controls included pEGFP-c1-transfected cells and HVS-GFP-infected cells. Expression of iPSC transgenes is confirmed by the presence of specific bands at 39, 23, and 34 kDa for Oct4, Lin28, and Nanog, respectively.

    Techniques Used: BAC Assay, Recombinant, Clone Assay, Polymerase Chain Reaction, Amplification, Construct, Expressing, Plasmid Preparation, Subcloning, Transfection, Infection

    9) Product Images from "The Ebola Virus VP35 Protein Inhibits Activation of Interferon Regulatory Factor 3"

    Article Title: The Ebola Virus VP35 Protein Inhibits Activation of Interferon Regulatory Factor 3

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.14.7945-7956.2003

    The Ebola virus VP35 protein prevents the nuclear translocation of hIRF-3 after SeV infection. (A) Fluorescence images showing expression of HA-tagged VP35(R) protein (red) and the corresponding distribution of GFP-hIRF-3 (green). Cells expressing both HA-tagged VP35 and GFP-IRF-3 are indicated by the large white arrows. Examples of cells with nuclear GFP-IRF-3 are indicated by the small yellow arrows. Vero cells were transfected with 0.4 μg of VP35(R) expression plasmid plus 0.8 μg of pEGFP-C1-hIRF3 and infected 24 h later with SeV. Eight hours postinfection, cells were fixed and stained with anti-HA monoclonal antibody (red). (B) The percentage of GFP-IRF-3-expressing cells with nuclear GFP-IRF-3 in cells transfected with the indicated plasmids and either mock infected or infected with SeV is shown. Vero cells were transfected with 0.4 μg of empty vector or expression plasmids for Ebola virus Zaire VP35 [VP35(Z)], HA-tagged Ebola virus Reston VP35 [HAVP35(R)], influenza virus NS1 protein (NS1), or Ebola virus Zaire virus VP24 protein (VP24), plus 0.2 μg of pEGFP-C1-hIRF3. At 24 h posttransfection, the cells were mock infected or infected with SeV at an MOI of 10. Eight hours postinfection, the cells were examined for GFP localization. The results are the average of two independent experiments where 200 to 300 cells were counted per transfection.
    Figure Legend Snippet: The Ebola virus VP35 protein prevents the nuclear translocation of hIRF-3 after SeV infection. (A) Fluorescence images showing expression of HA-tagged VP35(R) protein (red) and the corresponding distribution of GFP-hIRF-3 (green). Cells expressing both HA-tagged VP35 and GFP-IRF-3 are indicated by the large white arrows. Examples of cells with nuclear GFP-IRF-3 are indicated by the small yellow arrows. Vero cells were transfected with 0.4 μg of VP35(R) expression plasmid plus 0.8 μg of pEGFP-C1-hIRF3 and infected 24 h later with SeV. Eight hours postinfection, cells were fixed and stained with anti-HA monoclonal antibody (red). (B) The percentage of GFP-IRF-3-expressing cells with nuclear GFP-IRF-3 in cells transfected with the indicated plasmids and either mock infected or infected with SeV is shown. Vero cells were transfected with 0.4 μg of empty vector or expression plasmids for Ebola virus Zaire VP35 [VP35(Z)], HA-tagged Ebola virus Reston VP35 [HAVP35(R)], influenza virus NS1 protein (NS1), or Ebola virus Zaire virus VP24 protein (VP24), plus 0.2 μg of pEGFP-C1-hIRF3. At 24 h posttransfection, the cells were mock infected or infected with SeV at an MOI of 10. Eight hours postinfection, the cells were examined for GFP localization. The results are the average of two independent experiments where 200 to 300 cells were counted per transfection.

    Techniques Used: Translocation Assay, Infection, Fluorescence, Expressing, Transfection, Plasmid Preparation, Staining

    10) Product Images from "Development of a 2-Nitrobenzoate-Sensing Bioreporter Based on an Inducible Gene Cluster"

    Article Title: Development of a 2-Nitrobenzoate-Sensing Bioreporter Based on an Inducible Gene Cluster

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00254

    Construction of 2NBA bioreporter strains. (A) Genetic organization of the 2NBA degrading onb gene cluster in Cupriavidus sp. strain ST-14. Black bars indicate the genomic positions of the amplified Up and Down fragments used to construct the recombination plasmids. (B) The reporter gene EGFP was PCR amplified using pEGFP-C1 plasmid as template. (C) pCM184 was used as the vector backbone in which the Up1, EGFP and Down1 fragments were cloned in the depicted orientation to construct pCM-prox. The pCM-dist plasmid was constructed similarly using Up2, EGFP and Down2 fragments. (D) Recombination events occurred between pCM-prox and the ST-14 chromosome with the insertion of EGFP/Kan cassette in the place of onbFCA segment to construct the bioreporter strain BR prox . (E) Recombination between pCM-dist and ST-14 chromosome resulted in the construction of the bioreporter strain BR dist by disrupting the onbIGDB segment.
    Figure Legend Snippet: Construction of 2NBA bioreporter strains. (A) Genetic organization of the 2NBA degrading onb gene cluster in Cupriavidus sp. strain ST-14. Black bars indicate the genomic positions of the amplified Up and Down fragments used to construct the recombination plasmids. (B) The reporter gene EGFP was PCR amplified using pEGFP-C1 plasmid as template. (C) pCM184 was used as the vector backbone in which the Up1, EGFP and Down1 fragments were cloned in the depicted orientation to construct pCM-prox. The pCM-dist plasmid was constructed similarly using Up2, EGFP and Down2 fragments. (D) Recombination events occurred between pCM-prox and the ST-14 chromosome with the insertion of EGFP/Kan cassette in the place of onbFCA segment to construct the bioreporter strain BR prox . (E) Recombination between pCM-dist and ST-14 chromosome resulted in the construction of the bioreporter strain BR dist by disrupting the onbIGDB segment.

    Techniques Used: Amplification, Construct, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay

    11) Product Images from "Caspase-Dependent Regulation and Subcellular Redistribution of the Transcriptional Modulator YY1 during Apoptosis"

    Article Title: Caspase-Dependent Regulation and Subcellular Redistribution of the Transcriptional Modulator YY1 during Apoptosis

    Journal:

    doi: 10.1128/MCB.25.9.3704-3714.2005

    Enhancement of apoptosis by YY1Δ119. (A) HeLa cells were cotransfected with pEGFP-C1 (0.05 μg) in order to detect the transfected cells, an expression plasmid for the death receptor Fas (pCR3-Fas; 0.1 μg), and plasmids expressing
    Figure Legend Snippet: Enhancement of apoptosis by YY1Δ119. (A) HeLa cells were cotransfected with pEGFP-C1 (0.05 μg) in order to detect the transfected cells, an expression plasmid for the death receptor Fas (pCR3-Fas; 0.1 μg), and plasmids expressing

    Techniques Used: Transfection, Expressing, Plasmid Preparation

    12) Product Images from "The landscape of BRAF transcript and protein variants in human cancer"

    Article Title: The landscape of BRAF transcript and protein variants in human cancer

    Journal: Molecular Cancer

    doi: 10.1186/s12943-017-0645-4

    The X2 isoform displays a faster decay due to increased proteosomal-mediated degradation. a Schematic representation of the chimerical protein derived from the fusion of EGFP coding sequence with the CR3 domain of BRAFV600E-ref, X1, and X2 within the pEGFP-C1 plasmid. The asterisk indicates the presence of the V600E mutation. b - e Upon the transient transfection of pEGFP-C1 empty (pEGFP-empty), pEGFP-CR3-ref, pEGFP-CR3-X1, and pEGFP-CR3-X2 plasmids in A375 cells, real-time PCR performed with primers for EGFP and for total BRAF indicates that the chimerical mRNAs are all transcribed at similar levels ( b ), but western blot ( c ), flow cytometry ( d ) and confocal microscopy analysis ( e ) indicate that, when fused with CR3-X2, EGFP protein is expressed at lower levels. The dotted box shows a higher exposure of the anti-EGFP antibody. f - h When PIG-BRAFV600E-ΔCterm plasmid, which lacks the nucleotides encoding for the X2-specific C-terminal domain ( f ), is transiently transfected in HEK293T cells, not only BRAF mRNA ( g ), but also BRAF protein is detectable ( h ). e: empty PIG-NotI; X1: PIG-BRAFV600E-X1 (used as positive control); X2: PIG-BRAFV600E-X2; ΔCterm: PIG-BRAFV600E-ΔCterm. i - j Upon the transient transfection of pEGFP-empty, pEGFP-CR3-ref, pEGFP-CR3-X1, and pEGFP-CR3-X2 plasmids, A375 cells were treated with 100 ug/ml cicloheximide (CHX) ( i ) or 20 uM MG132 ( j ) for 8 h. The CHX treatment indicates that the decay rate of CR3-X2 is faster than that of CR3-ref and CR3-X1, while the MG132 treatment suggests that this is due to higher degradation rate through the ubiquitin-proteasome pathway. k The prediction of potential proteasomal cleavage sites using 3 different algorithms retrieves the indicated X2-specific consensus peptide. ( l ) The mutagenesis of Lys739 into a proteasome-insensitive Arg rescues the expression of the X2 isoform of BRAF protein. e: empty PIG-NotI; X1: PIG-BRAFV600E-X1 (used as positive control); X2: PIG-BRAFV600E-X2; X2 K739R : PIG-BRAFV600E-X2 in which Lys(K)739 has been substituted with Arg(R) (AAA to AGA triplet change). m Cartoon that summarizes the main findings of this article (details in the text). The pictures are taken from 1 out of 3 independent experiments performed, all with comparable outcome. The graphs represent the mean ± SEM of 3 independent experiments. * p
    Figure Legend Snippet: The X2 isoform displays a faster decay due to increased proteosomal-mediated degradation. a Schematic representation of the chimerical protein derived from the fusion of EGFP coding sequence with the CR3 domain of BRAFV600E-ref, X1, and X2 within the pEGFP-C1 plasmid. The asterisk indicates the presence of the V600E mutation. b - e Upon the transient transfection of pEGFP-C1 empty (pEGFP-empty), pEGFP-CR3-ref, pEGFP-CR3-X1, and pEGFP-CR3-X2 plasmids in A375 cells, real-time PCR performed with primers for EGFP and for total BRAF indicates that the chimerical mRNAs are all transcribed at similar levels ( b ), but western blot ( c ), flow cytometry ( d ) and confocal microscopy analysis ( e ) indicate that, when fused with CR3-X2, EGFP protein is expressed at lower levels. The dotted box shows a higher exposure of the anti-EGFP antibody. f - h When PIG-BRAFV600E-ΔCterm plasmid, which lacks the nucleotides encoding for the X2-specific C-terminal domain ( f ), is transiently transfected in HEK293T cells, not only BRAF mRNA ( g ), but also BRAF protein is detectable ( h ). e: empty PIG-NotI; X1: PIG-BRAFV600E-X1 (used as positive control); X2: PIG-BRAFV600E-X2; ΔCterm: PIG-BRAFV600E-ΔCterm. i - j Upon the transient transfection of pEGFP-empty, pEGFP-CR3-ref, pEGFP-CR3-X1, and pEGFP-CR3-X2 plasmids, A375 cells were treated with 100 ug/ml cicloheximide (CHX) ( i ) or 20 uM MG132 ( j ) for 8 h. The CHX treatment indicates that the decay rate of CR3-X2 is faster than that of CR3-ref and CR3-X1, while the MG132 treatment suggests that this is due to higher degradation rate through the ubiquitin-proteasome pathway. k The prediction of potential proteasomal cleavage sites using 3 different algorithms retrieves the indicated X2-specific consensus peptide. ( l ) The mutagenesis of Lys739 into a proteasome-insensitive Arg rescues the expression of the X2 isoform of BRAF protein. e: empty PIG-NotI; X1: PIG-BRAFV600E-X1 (used as positive control); X2: PIG-BRAFV600E-X2; X2 K739R : PIG-BRAFV600E-X2 in which Lys(K)739 has been substituted with Arg(R) (AAA to AGA triplet change). m Cartoon that summarizes the main findings of this article (details in the text). The pictures are taken from 1 out of 3 independent experiments performed, all with comparable outcome. The graphs represent the mean ± SEM of 3 independent experiments. * p

    Techniques Used: Derivative Assay, Sequencing, Plasmid Preparation, Mutagenesis, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry, Cytometry, Confocal Microscopy, Positive Control, Expressing

    13) Product Images from "Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps"

    Article Title: Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198203

    Overexpression of XIAP or XIAP subdomains hardly affect TRAIL sensitivity. (A) and (B) Transient transfection of XIAP and XIAP-derived BIR domains. NCI-H460 cells were transiently transfected with empty vector (pEGFP-C1) as a control or vectors encoding full length XIAP or the respective BIR2 and BIR3 domain. Transfection efficiency was checked by visualizing the GFP signal microscopically after 24 h (A). Whole cell lysates were run on a SDS-PAGE and immunoblot analysis was performed (B) using anti-GFP antibody. (C) Cells from (A) were stimulated with serial dilutions of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and the absorbance was determined at 550 nm. Cells transfected with pEGFP-C1 were used as control. Shown are mean values ± SD calculated from triplicates. One representative experiment out of three is shown.
    Figure Legend Snippet: Overexpression of XIAP or XIAP subdomains hardly affect TRAIL sensitivity. (A) and (B) Transient transfection of XIAP and XIAP-derived BIR domains. NCI-H460 cells were transiently transfected with empty vector (pEGFP-C1) as a control or vectors encoding full length XIAP or the respective BIR2 and BIR3 domain. Transfection efficiency was checked by visualizing the GFP signal microscopically after 24 h (A). Whole cell lysates were run on a SDS-PAGE and immunoblot analysis was performed (B) using anti-GFP antibody. (C) Cells from (A) were stimulated with serial dilutions of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and the absorbance was determined at 550 nm. Cells transfected with pEGFP-C1 were used as control. Shown are mean values ± SD calculated from triplicates. One representative experiment out of three is shown.

    Techniques Used: Over Expression, Transfection, Derivative Assay, Plasmid Preparation, SDS Page, Staining

    14) Product Images from "Functional Cooperation of Epstein-Barr Virus Nuclear Antigen 2 and the Survival Motor Neuron Protein in Transactivation of the Viral LMP1 Promoter"

    Article Title: Functional Cooperation of Epstein-Barr Virus Nuclear Antigen 2 and the Survival Motor Neuron Protein in Transactivation of the Viral LMP1 Promoter

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.23.11781-11790.2001

    (A) Interaction of DP103 and SMN in B lymphocytes. Coimmunoprecipitations (IP) from Raji cell extracts were performed with DP103-specific MAb 9A3 (IP: DP103 Ab) or an irrelevant control antibody (anti-TrypE 3A6, IP: control Ab), followed by SDS–10% PAGE and Western blotting. Precipitated proteins were detected with anti-SMN-N serum (Santa Cruz Biochemicals) (left panel, WB: anti SMN) or anti-DP103 MAb 8H4 (right panel, WB: anti DP103). The positions of SMN and DP103 are indicated by arrows. Lanes designated Raji input represent ca. 1% of unprecipitated Raji cell extract. The positions of the molecular mass marker proteins are indicated on the left side (in kilodaltons). (B) SMN coactivates the viral LMP1 promoter in the presence of EBNA2. BJAB cells were transfected with luciferase reporter constructs encoding positions −327/+40 (EBNA2 responsive) or −154/+40 (nonresponsive) of the LMP1 promoter (4 μg) and the indicated combinations of pSG5 constructs encoding EBNA2 or HA-tagged SMN and DP103 (10 μg). After 48 h, the cells were lysed by freeze-thawing, and the luciferase activity was measured. The transfection efficiency was determined by scanning the expression of cotransfected pEGFP-C1 vector (2 μg) by FACS analysis prior to lysis of the cells. For each experiment, luciferase values standardized for transfection efficiency were calculated relative to the values obtained by EBNA2 and the respective full-length promoter construct (set to 100%). Graphs represent the mean values of five independent experiments (± the standard error of the mean [SEM]).
    Figure Legend Snippet: (A) Interaction of DP103 and SMN in B lymphocytes. Coimmunoprecipitations (IP) from Raji cell extracts were performed with DP103-specific MAb 9A3 (IP: DP103 Ab) or an irrelevant control antibody (anti-TrypE 3A6, IP: control Ab), followed by SDS–10% PAGE and Western blotting. Precipitated proteins were detected with anti-SMN-N serum (Santa Cruz Biochemicals) (left panel, WB: anti SMN) or anti-DP103 MAb 8H4 (right panel, WB: anti DP103). The positions of SMN and DP103 are indicated by arrows. Lanes designated Raji input represent ca. 1% of unprecipitated Raji cell extract. The positions of the molecular mass marker proteins are indicated on the left side (in kilodaltons). (B) SMN coactivates the viral LMP1 promoter in the presence of EBNA2. BJAB cells were transfected with luciferase reporter constructs encoding positions −327/+40 (EBNA2 responsive) or −154/+40 (nonresponsive) of the LMP1 promoter (4 μg) and the indicated combinations of pSG5 constructs encoding EBNA2 or HA-tagged SMN and DP103 (10 μg). After 48 h, the cells were lysed by freeze-thawing, and the luciferase activity was measured. The transfection efficiency was determined by scanning the expression of cotransfected pEGFP-C1 vector (2 μg) by FACS analysis prior to lysis of the cells. For each experiment, luciferase values standardized for transfection efficiency were calculated relative to the values obtained by EBNA2 and the respective full-length promoter construct (set to 100%). Graphs represent the mean values of five independent experiments (± the standard error of the mean [SEM]).

    Techniques Used: Polyacrylamide Gel Electrophoresis, Western Blot, Marker, Transfection, Luciferase, Construct, Activity Assay, Expressing, Plasmid Preparation, FACS, Lysis

    15) Product Images from "Localization and Characterization of Rat Transmembrane Protein 225 Specifically Expressed in Testis"

    Article Title: Localization and Characterization of Rat Transmembrane Protein 225 Specifically Expressed in Testis

    Journal: DNA and Cell Biology

    doi: 10.1089/dna.2010.1048

    Subcellular localization of TMEM225 protein in HeLa cells. (A–C) Cells transfected with pEGFP-C1. (D–F) Cells transfected with pEGFP-TMEM225. (G–I) Cells transfected with pEGFP-D35-TMEM225. (A, D, G) The nucleus of cells were stained
    Figure Legend Snippet: Subcellular localization of TMEM225 protein in HeLa cells. (A–C) Cells transfected with pEGFP-C1. (D–F) Cells transfected with pEGFP-TMEM225. (G–I) Cells transfected with pEGFP-D35-TMEM225. (A, D, G) The nucleus of cells were stained

    Techniques Used: Transfection, Staining

    16) Product Images from "Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids"

    Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043283

    Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
    Figure Legend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

    Techniques Used: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

    Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.
    Figure Legend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

    Techniques Used: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

    17) Product Images from "Capacity to Elicit Cytotoxic CD8 T Cell Activity Against Mycobacterium avium subsp. paratuberculosis Is Retained in a Vaccine Candidate 35 kDa Peptide Modified for Expression in Mammalian Cells"

    Article Title: Capacity to Elicit Cytotoxic CD8 T Cell Activity Against Mycobacterium avium subsp. paratuberculosis Is Retained in a Vaccine Candidate 35 kDa Peptide Modified for Expression in Mammalian Cells

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02859

    Quantitative Western immunoblot of serum-free medium containing secreted tPA-MMP-5mut, tPA-MMP-2mut, and tPA-MMP from pCMV-tPA-MMP-5mut, pEF1α-tPA-MMP-5mut, pCMV-tPA-MMP-2mut, pEF1α-tPA-MMP-2mut, pCMV-tPA-MMP, and pEF1α-tPA-MMP transfected HEK 293T cells at 48 h post transfection. Each lane was loaded with 15 μl of serum-free medium supernatant. The same number of HEK 293T cells were transfected with the same amount of each DNA plasmid with the same efficiency of transfection (100%), as determined by co-transfection with pEGFP-C1. Band intensity was quantified using densitometry (ChemiDoc; MPIMAGING SYSTEM, LAB SOFTWARE, BioRad), and displayed using a bar graph. Data were normalized as folds of increased secretion, where pEF1α-tPA-MMP-5mut signal was considered equal to 1. The experiment was repeated three times, and statistical significance ( P
    Figure Legend Snippet: Quantitative Western immunoblot of serum-free medium containing secreted tPA-MMP-5mut, tPA-MMP-2mut, and tPA-MMP from pCMV-tPA-MMP-5mut, pEF1α-tPA-MMP-5mut, pCMV-tPA-MMP-2mut, pEF1α-tPA-MMP-2mut, pCMV-tPA-MMP, and pEF1α-tPA-MMP transfected HEK 293T cells at 48 h post transfection. Each lane was loaded with 15 μl of serum-free medium supernatant. The same number of HEK 293T cells were transfected with the same amount of each DNA plasmid with the same efficiency of transfection (100%), as determined by co-transfection with pEGFP-C1. Band intensity was quantified using densitometry (ChemiDoc; MPIMAGING SYSTEM, LAB SOFTWARE, BioRad), and displayed using a bar graph. Data were normalized as folds of increased secretion, where pEF1α-tPA-MMP-5mut signal was considered equal to 1. The experiment was repeated three times, and statistical significance ( P

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Cotransfection, Software

    (A) Diagram of a third-generation lentiviral transfer vector delivering tPA-MMP ORF (tPAp35AU1, green) expression cassette and GFP/Puromycin reporter gene/selectable marker (EGFP:T2A:Puro, purple) expression cassette. Different genetic elements depicted within the construct from nucleotide position 0 are: RSV promoter (blue violet); Δ5′ LTR (brown); Ψ (medium blue); RRE (chocolate); cPPT (brown); EF1α (navy); WPRE (royal blue); CMV promoter (orchid); ΔU3/3' LTR (sienna); Sv40 early pA (red); Ampicillin (turquoise); pUC ori (dark salmon). (B) Western immunoblot of pEF1α-tPA-MMP and pEGFP-C1 transfected HEK 293T cell extracts and supernatant. Lanes with cell extract were loaded with 20 μg of total protein, whereas the lanes with supernatant were loaded with 20 μl of serum-free medium incubated for 48 h with transfected cells. Cells transfected with pEGFPC-1 served as negative controls ( Mock ). (C) Western immunoblot of pEF1a-tPA-MMP-CMV-EGFP:T2A:Puro-transduced and Puromicin-selected HKB-11 cells, supernatant collected at 4, 8, 24, and 48 h incubation. In each lane, 15 μl of conditioned serum-free medium was loaded. The negative control (–) was established with a similar lentiviral vector delivering only GFP (pEF1α-iresGFP).
    Figure Legend Snippet: (A) Diagram of a third-generation lentiviral transfer vector delivering tPA-MMP ORF (tPAp35AU1, green) expression cassette and GFP/Puromycin reporter gene/selectable marker (EGFP:T2A:Puro, purple) expression cassette. Different genetic elements depicted within the construct from nucleotide position 0 are: RSV promoter (blue violet); Δ5′ LTR (brown); Ψ (medium blue); RRE (chocolate); cPPT (brown); EF1α (navy); WPRE (royal blue); CMV promoter (orchid); ΔU3/3' LTR (sienna); Sv40 early pA (red); Ampicillin (turquoise); pUC ori (dark salmon). (B) Western immunoblot of pEF1α-tPA-MMP and pEGFP-C1 transfected HEK 293T cell extracts and supernatant. Lanes with cell extract were loaded with 20 μg of total protein, whereas the lanes with supernatant were loaded with 20 μl of serum-free medium incubated for 48 h with transfected cells. Cells transfected with pEGFPC-1 served as negative controls ( Mock ). (C) Western immunoblot of pEF1a-tPA-MMP-CMV-EGFP:T2A:Puro-transduced and Puromicin-selected HKB-11 cells, supernatant collected at 4, 8, 24, and 48 h incubation. In each lane, 15 μl of conditioned serum-free medium was loaded. The negative control (–) was established with a similar lentiviral vector delivering only GFP (pEF1α-iresGFP).

    Techniques Used: Plasmid Preparation, Expressing, Marker, Construct, Western Blot, Transfection, Incubation, Negative Control

    (A) MMP N-linked (1N, 2N, and 3N in red) and O-linked (1T and 2T in blue) predicted glycosylation sites. (B) Western immunoblot of tPA-MMP secreted protein treated (Sup PNGase treated) or untreated (Sup untreated) with PGNase F. Cell extracts from pEF1α-tPA-MMP (Cell extract) and pEGFP-C1 (Cell extract GFP) transfected HEK 293T cells were used as positive and negative controls, respectively.
    Figure Legend Snippet: (A) MMP N-linked (1N, 2N, and 3N in red) and O-linked (1T and 2T in blue) predicted glycosylation sites. (B) Western immunoblot of tPA-MMP secreted protein treated (Sup PNGase treated) or untreated (Sup untreated) with PGNase F. Cell extracts from pEF1α-tPA-MMP (Cell extract) and pEGFP-C1 (Cell extract GFP) transfected HEK 293T cells were used as positive and negative controls, respectively.

    Techniques Used: Western Blot, Transfection

    18) Product Images from "Dynamics of Actin-Based Movement by Rickettsia rickettsii in Vero Cells"

    Article Title: Dynamics of Actin-Based Movement by Rickettsia rickettsii in Vero Cells

    Journal: Infection and Immunity

    doi:

    Time-lapse video fluorescence microscopy of Rickettsia ABM documenting the stationary nature of intracytoplasmic rickettsial actin tails. Vero cells were transfected with the plasmid pEGFP-C1/actin encoding GFP–β-actin and infected with R. rickettsii . Laser-scanning confocal fluorescence microscopy was conducted with live cells. Images were collected every 21 s. Gaps (outlined areas) that arise because of the helical twisting of the bifurcated rickettsial tail appendage remain stationary as the bacterium moves forward. Bar, 2 μm.
    Figure Legend Snippet: Time-lapse video fluorescence microscopy of Rickettsia ABM documenting the stationary nature of intracytoplasmic rickettsial actin tails. Vero cells were transfected with the plasmid pEGFP-C1/actin encoding GFP–β-actin and infected with R. rickettsii . Laser-scanning confocal fluorescence microscopy was conducted with live cells. Images were collected every 21 s. Gaps (outlined areas) that arise because of the helical twisting of the bifurcated rickettsial tail appendage remain stationary as the bacterium moves forward. Bar, 2 μm.

    Techniques Used: Fluorescence, Microscopy, Transfection, Plasmid Preparation, Infection

    Time-lapse video fluorescence microscopy of Listeria and Rickettsia ABM. Vero cells were transfected with the plasmid pEGFP-C1/actin encoding GFP–β-actin and infected with either L. monocytogenes or R. rickettsii . Laser-scanning confocal fluorescence microscopy was conducted with live cells, and images were collected every 21 s. (A) Actin-based movement of Listeria . The average rate of movement was 12 μm/min ( n = 23). (B) Actin-based movement of Rickettsia . The average rate of movement was 4.8 μm/min ( n = 28). Bars, 2.5 μm.
    Figure Legend Snippet: Time-lapse video fluorescence microscopy of Listeria and Rickettsia ABM. Vero cells were transfected with the plasmid pEGFP-C1/actin encoding GFP–β-actin and infected with either L. monocytogenes or R. rickettsii . Laser-scanning confocal fluorescence microscopy was conducted with live cells, and images were collected every 21 s. (A) Actin-based movement of Listeria . The average rate of movement was 12 μm/min ( n = 23). (B) Actin-based movement of Rickettsia . The average rate of movement was 4.8 μm/min ( n = 28). Bars, 2.5 μm.

    Techniques Used: Fluorescence, Microscopy, Transfection, Plasmid Preparation, Infection

    Incorporation of GFP-actin into COS-7 cell stress fibers and actin tails of R. rickettsii and Listeria monocytogenes within Vero cells. Mammalian cell cultures were transfected with the plasmid pEGFP-C1/actin encoding GFP–β-actin and infected with either L. monocytogenes or R. rickettsii . Transfected COS-7 cells were fixed and viewed without subsequent staining. Infected Vero cells were fixed and permeabilized, and intracellular bacteria were stained by indirect immunofluorescence with the second antibody conjugated to rhodamine (red). (A) Incorporation of GFP-actin (green) into stress fibers of COS-7 cells. (B) Incorporation of GFP-actin into R. rickettsii actin tails. Note the cluster of organisms undergoing binary fission with one large polar actin tail. (C) Incorporation of GFP-actin into L. monocytogenes actin tails. Bars: panel A, 15 μm; panel B, 2 μm; panel C, 3 μm.
    Figure Legend Snippet: Incorporation of GFP-actin into COS-7 cell stress fibers and actin tails of R. rickettsii and Listeria monocytogenes within Vero cells. Mammalian cell cultures were transfected with the plasmid pEGFP-C1/actin encoding GFP–β-actin and infected with either L. monocytogenes or R. rickettsii . Transfected COS-7 cells were fixed and viewed without subsequent staining. Infected Vero cells were fixed and permeabilized, and intracellular bacteria were stained by indirect immunofluorescence with the second antibody conjugated to rhodamine (red). (A) Incorporation of GFP-actin (green) into stress fibers of COS-7 cells. (B) Incorporation of GFP-actin into R. rickettsii actin tails. Note the cluster of organisms undergoing binary fission with one large polar actin tail. (C) Incorporation of GFP-actin into L. monocytogenes actin tails. Bars: panel A, 15 μm; panel B, 2 μm; panel C, 3 μm.

    Techniques Used: Transfection, Plasmid Preparation, Infection, Staining, Immunofluorescence

    Time-lapse video fluorescence microscopy of intranuclear R. rickettsii . Vero cells were transfected with the plasmid pEGFP-C1/actin encoding a GFP–β-actin chimera. Cells were subsequently infected with R. rickettsii . Laser-scanning confocal fluorescence microscopy was conducted with live cells, and images were collected every 21 s. Two adjacent transfected cells are depicted in this micrograph. The strongly fluorescing cytoplasm surrounds the darker, oval-shaped nucleus. This 63-s sequence demonstrates force being exerted against the nuclear envelope by both the cluster of rickettsiae at the head of the actin tail (arrow) and an interior portion of the actin tail. Dramatic movement of the middle of the actin tail is obvious, with no forward movement of rickettsiae observed. The end of the actin tail is visible at the bottom right of the nucleus. Bar, 5 μm.
    Figure Legend Snippet: Time-lapse video fluorescence microscopy of intranuclear R. rickettsii . Vero cells were transfected with the plasmid pEGFP-C1/actin encoding a GFP–β-actin chimera. Cells were subsequently infected with R. rickettsii . Laser-scanning confocal fluorescence microscopy was conducted with live cells, and images were collected every 21 s. Two adjacent transfected cells are depicted in this micrograph. The strongly fluorescing cytoplasm surrounds the darker, oval-shaped nucleus. This 63-s sequence demonstrates force being exerted against the nuclear envelope by both the cluster of rickettsiae at the head of the actin tail (arrow) and an interior portion of the actin tail. Dramatic movement of the middle of the actin tail is obvious, with no forward movement of rickettsiae observed. The end of the actin tail is visible at the bottom right of the nucleus. Bar, 5 μm.

    Techniques Used: Fluorescence, Microscopy, Transfection, Plasmid Preparation, Infection, Sequencing

    19) Product Images from "Epstein-Barr Virus BALF3 Has Nuclease Activity and Mediates Mature Virion Production during the Lytic Cycle"

    Article Title: Epstein-Barr Virus BALF3 Has Nuclease Activity and Mediates Mature Virion Production during the Lytic Cycle

    Journal: Journal of Virology

    doi: 10.1128/JVI.00063-14

    Subcellular localization of EBV BALF3 in EBV-positive cells during the lytic cycle. TW01 and NA cells were transiently transfected with pEGFP-C1-BALF3, followed by induction with 40 ng/ml TPA and 3 mM SB for 36 h, and the images shown were obtained by indirect immunofluorescence staining and photographed using a fluorescence microscope (A) and a confocal microscope (B). The detection of EBV BMRF1 with the specific antibody indicates lytic cycle activation, and the nuclei of the cells were stained with Hoechst 33258. (C) Protein extracts of cells expressing GFP-BALF3 were separated into input (I), cytosolic (C), and nuclear (N) fractions and subjected to Western blotting. After electrophoresis, GFP-BALF3 was detected by an antibody specific to GFP, and PARP1 and α-tubulin were used as markers for the nucleus and the cytoplasm, respectively.
    Figure Legend Snippet: Subcellular localization of EBV BALF3 in EBV-positive cells during the lytic cycle. TW01 and NA cells were transiently transfected with pEGFP-C1-BALF3, followed by induction with 40 ng/ml TPA and 3 mM SB for 36 h, and the images shown were obtained by indirect immunofluorescence staining and photographed using a fluorescence microscope (A) and a confocal microscope (B). The detection of EBV BMRF1 with the specific antibody indicates lytic cycle activation, and the nuclei of the cells were stained with Hoechst 33258. (C) Protein extracts of cells expressing GFP-BALF3 were separated into input (I), cytosolic (C), and nuclear (N) fractions and subjected to Western blotting. After electrophoresis, GFP-BALF3 was detected by an antibody specific to GFP, and PARP1 and α-tubulin were used as markers for the nucleus and the cytoplasm, respectively.

    Techniques Used: Transfection, Immunofluorescence, Staining, Fluorescence, Microscopy, Activation Assay, Expressing, Western Blot, Electrophoresis

    20) Product Images from "The Open Reading Frame 57 Gene Product of Herpesvirus Saimiri Shuttles between the Nucleus and Cytoplasm and Is Involved in Viral RNA Nuclear Export"

    Article Title: The Open Reading Frame 57 Gene Product of Herpesvirus Saimiri Shuttles between the Nucleus and Cytoplasm and Is Involved in Viral RNA Nuclear Export

    Journal: Journal of Virology

    doi:

    The ORF 57 gene product expresses a nuclear export signal. Cos-7 cell monolayers were transfected with 2 μg of either pEGFP-C1 (i) or pEGFP-57NES (ii). After 24 h, the subcellular localization of GFP was observed by using fluorescence microscopy.
    Figure Legend Snippet: The ORF 57 gene product expresses a nuclear export signal. Cos-7 cell monolayers were transfected with 2 μg of either pEGFP-C1 (i) or pEGFP-57NES (ii). After 24 h, the subcellular localization of GFP was observed by using fluorescence microscopy.

    Techniques Used: Transfection, Fluorescence, Microscopy

    21) Product Images from "PIN-G - A novel reporter for imaging and defining the effects of trafficking signals in membrane proteins"

    Article Title: PIN-G - A novel reporter for imaging and defining the effects of trafficking signals in membrane proteins

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-6-15

    Cloning scheme for the preparation of the pIN-G construct and the transmembrane topology of the pIN-G reporter protein. 1A . A fragment encoding EGFP, obtained by AgeI/SalI digestion of pEGFP-C1, was ligated into an XmaI/SalI digested pDisplay backbone and the entire protein coding region PCR-amplified and ligated into a host backbone generated through NheI/KpnI digestion of pEGFP-C1 to retain the 3' multiple cloning site but exclude the EGFP coding region. The final pIN-G construct, with the appropriate reading frame throughout, was generated via a subsequent round of PCR. See Methods for further details. 1B . Following expression and cleavage of the Igk- chain leader sequence, the pIN-G protein is proposed to adopt a transmembrane orientation where HA and cMyc epitope tags and GFP have an extracellular disposition at the cell surface (or intra-lumenal in organelles or vesicular trafficking intermediates).
    Figure Legend Snippet: Cloning scheme for the preparation of the pIN-G construct and the transmembrane topology of the pIN-G reporter protein. 1A . A fragment encoding EGFP, obtained by AgeI/SalI digestion of pEGFP-C1, was ligated into an XmaI/SalI digested pDisplay backbone and the entire protein coding region PCR-amplified and ligated into a host backbone generated through NheI/KpnI digestion of pEGFP-C1 to retain the 3' multiple cloning site but exclude the EGFP coding region. The final pIN-G construct, with the appropriate reading frame throughout, was generated via a subsequent round of PCR. See Methods for further details. 1B . Following expression and cleavage of the Igk- chain leader sequence, the pIN-G protein is proposed to adopt a transmembrane orientation where HA and cMyc epitope tags and GFP have an extracellular disposition at the cell surface (or intra-lumenal in organelles or vesicular trafficking intermediates).

    Techniques Used: Clone Assay, Construct, Polymerase Chain Reaction, Amplification, Generated, Expressing, Sequencing

    22) Product Images from "Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps"

    Article Title: Bcl-2-mediated control of TRAIL-induced apoptotic response in the non-small lung cancer cell line NCI-H460 is effective at late caspase processing steps

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198203

    Overexpression of XIAP or XIAP subdomains hardly affect TRAIL sensitivity. (A) and (B) Transient transfection of XIAP and XIAP-derived BIR domains. NCI-H460 cells were transiently transfected with empty vector (pEGFP-C1) as a control or vectors encoding full length XIAP or the respective BIR2 and BIR3 domain. Transfection efficiency was checked by visualizing the GFP signal microscopically after 24 h (A). Whole cell lysates were run on a SDS-PAGE and immunoblot analysis was performed (B) using anti-GFP antibody. (C) Cells from (A) were stimulated with serial dilutions of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and the absorbance was determined at 550 nm. Cells transfected with pEGFP-C1 were used as control. Shown are mean values ± SD calculated from triplicates. One representative experiment out of three is shown.
    Figure Legend Snippet: Overexpression of XIAP or XIAP subdomains hardly affect TRAIL sensitivity. (A) and (B) Transient transfection of XIAP and XIAP-derived BIR domains. NCI-H460 cells were transiently transfected with empty vector (pEGFP-C1) as a control or vectors encoding full length XIAP or the respective BIR2 and BIR3 domain. Transfection efficiency was checked by visualizing the GFP signal microscopically after 24 h (A). Whole cell lysates were run on a SDS-PAGE and immunoblot analysis was performed (B) using anti-GFP antibody. (C) Cells from (A) were stimulated with serial dilutions of Db-scTRAIL for 24 h. Viable cells were stained with crystal violet and the absorbance was determined at 550 nm. Cells transfected with pEGFP-C1 were used as control. Shown are mean values ± SD calculated from triplicates. One representative experiment out of three is shown.

    Techniques Used: Over Expression, Transfection, Derivative Assay, Plasmid Preparation, SDS Page, Staining

    23) Product Images from "Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids"

    Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043283

    Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
    Figure Legend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

    Techniques Used: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

    Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.
    Figure Legend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

    Techniques Used: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

    24) Product Images from "Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids"

    Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043283

    Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
    Figure Legend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

    Techniques Used: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

    Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.
    Figure Legend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

    Techniques Used: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

    25) Product Images from "Serine Carboxypeptidase SCPEP1 and Cathepsin A Play Complementary Roles in Regulation of Vasoconstriction via Inactivation of Endothelin-1"

    Article Title: Serine Carboxypeptidase SCPEP1 and Cathepsin A Play Complementary Roles in Regulation of Vasoconstriction via Inactivation of Endothelin-1

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004146

    Scpep1 has carboxypeptidase activity against ET-1. A. Carboxypeptidase activity against ET-1 in homogenates of cultured AVSMC from WT, Scpep1 −/− , CathA S190A and Scpep1 −/− /CathA S190A mice as well as from the cells of Scpep1 −/− /CathA S190A mice transiently transfected with pCMV-GFP and pCMV-Scpep1 plasmids. AVSMC after the passage 3 were transfected or not with Scpep1-RGS-His-Tag and pEGFP-C1 plasmids. Forty-eight hours after transfection confluent cells were harvested, homogenized and tested for carboxypeptidase activity using 50 µM ET-1 as a substrate. Data are expressed as means (±S.D.) of 3 independent experiments performed with different cell cultures. * p
    Figure Legend Snippet: Scpep1 has carboxypeptidase activity against ET-1. A. Carboxypeptidase activity against ET-1 in homogenates of cultured AVSMC from WT, Scpep1 −/− , CathA S190A and Scpep1 −/− /CathA S190A mice as well as from the cells of Scpep1 −/− /CathA S190A mice transiently transfected with pCMV-GFP and pCMV-Scpep1 plasmids. AVSMC after the passage 3 were transfected or not with Scpep1-RGS-His-Tag and pEGFP-C1 plasmids. Forty-eight hours after transfection confluent cells were harvested, homogenized and tested for carboxypeptidase activity using 50 µM ET-1 as a substrate. Data are expressed as means (±S.D.) of 3 independent experiments performed with different cell cultures. * p

    Techniques Used: Activity Assay, Cell Culture, Mouse Assay, Transfection

    26) Product Images from "The identification of FANCD2 DNA binding domains reveals nuclear localization sequences"

    Article Title: The identification of FANCD2 DNA binding domains reveals nuclear localization sequences

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx543

    DNA binding residues in region F1 acts as a Nuclear Localization Signal for FANCD2. ( A ) Represents the hit map of the putative NLS sequences using the NLS prediction tool ( www.moseslab.csb.utoronto.ca/NLStradamus ) over full length FANCD2 and FANCI. ( B ) FANCD2-deficient cells (FA-D2) were transfected with the indicated citrine tagged FANCD2 expression constructs. Twenty-four hours post transfection, cells were fixed and stained with 4,6-diaminidino-2-phenyllindole (DAPI) to mark the nucleus. ( C ) A total of 60 cells transfected with the citrine vector, FANCD2 WT, F1Mut, F3Mut, F1+F3Mut and were scored and box plotted for percentage of citrine expressed in the nucleus. ( D ) NLS sequences used for F1 and FANCI. ( E ) NLS sequences predicted in (A) were fused to eGFP and transfected in HeLa cells. peGFP-C1 and SYT1 were used as vector and a cytoplasmic control. Twenty-four hours post transfection, cells were fixed and stained with 4,6-diaminidino-2-phenyllindole (DAPI) to mark the nucleus. Representative images of each expression constructs are shown. ( F ) A total of 60 cells were counted for F1NLS, FANCI NLS, pEGFP-C1, and SYT1 were scored and box plotted for percentage of eGFP expressed in the nucleus. Wilcoxon's test was employed, P -value
    Figure Legend Snippet: DNA binding residues in region F1 acts as a Nuclear Localization Signal for FANCD2. ( A ) Represents the hit map of the putative NLS sequences using the NLS prediction tool ( www.moseslab.csb.utoronto.ca/NLStradamus ) over full length FANCD2 and FANCI. ( B ) FANCD2-deficient cells (FA-D2) were transfected with the indicated citrine tagged FANCD2 expression constructs. Twenty-four hours post transfection, cells were fixed and stained with 4,6-diaminidino-2-phenyllindole (DAPI) to mark the nucleus. ( C ) A total of 60 cells transfected with the citrine vector, FANCD2 WT, F1Mut, F3Mut, F1+F3Mut and were scored and box plotted for percentage of citrine expressed in the nucleus. ( D ) NLS sequences used for F1 and FANCI. ( E ) NLS sequences predicted in (A) were fused to eGFP and transfected in HeLa cells. peGFP-C1 and SYT1 were used as vector and a cytoplasmic control. Twenty-four hours post transfection, cells were fixed and stained with 4,6-diaminidino-2-phenyllindole (DAPI) to mark the nucleus. Representative images of each expression constructs are shown. ( F ) A total of 60 cells were counted for F1NLS, FANCI NLS, pEGFP-C1, and SYT1 were scored and box plotted for percentage of eGFP expressed in the nucleus. Wilcoxon's test was employed, P -value

    Techniques Used: Binding Assay, Transfection, Expressing, Construct, Staining, Plasmid Preparation

    27) Product Images from "SnoVectors for nuclear expression of RNA"

    Article Title: SnoVectors for nuclear expression of RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1050

    snoVectors can lead to the nuclear retention of different types of RNA sequences. ( A ) Different types of RNAs, including intron sequences, a lncRNA and mRNAs, expressed from pZW1-snoVector are retained in the nucleus. Total RNAs and fractionated nuclear and cytoplasmic RNAs were collected from HeLa cells transfected with the indicated plasmids individually and then resolved on an agarose gel. Transcripts of different types of expressed RNAs (indicated by white arrows) were probed with Dig-labeled antisense SNORD116-13 and their predicted sizes are labeled at the bottom of each NB. tRNA lys was used as markers for nuclear/cytoplasmic RNA isolation. Equal amounts of total, cytoplasmic and nuclear RNAs were loaded onto an agarose gel and rRNAs were used as the loading control. ( B ) Different types of RNAs, including intron sequences, a lncRNA and mRNAs, expressed from pEGFP-C1 are found in both the nucleus and in the cytoplasm. Total RNAs and fractionated nuclear and cytoplasmic RNAs were collected from HeLa cells transfected with the indicated plasmids individually and then resolved on an agarose gel. Transcripts of different types of expressed RNAs (indicated by white arrows) were probed with Dig-labeled egfp and their predicted sizes were labeled in the bottom of each NB. See (A) for details. ( C) Different types of RNAs expressed from pZW1-snoVector are retained in the nucleus, while those expressed from pEGFP-C1 are found in both the nucleus and the cytoplasm. ( D ) Different types of RNAs expressed from pcDNA3.0 vector are found in both the nucleus and the cytoplasm. In (A) and (B), white arrows indicate the expected RNAs expressed from different vectors. Assays were repeated and the same results were obtained. In (C) and (D), nuclear and cytoplasmic RNAs extracted from equal numbers of transfected HeLa cells under each indicated condition were assayed by RT-qPCR. The cytoplasmic distributed gapdh and the nuclear retained endogenous hNEAT1 were used as markers to indicate a qualified cytoplasmic and nuclear fractionation under each transfection condition. Error bars were calculated from three replicates. Also see supplemental information Figures S5–S6.
    Figure Legend Snippet: snoVectors can lead to the nuclear retention of different types of RNA sequences. ( A ) Different types of RNAs, including intron sequences, a lncRNA and mRNAs, expressed from pZW1-snoVector are retained in the nucleus. Total RNAs and fractionated nuclear and cytoplasmic RNAs were collected from HeLa cells transfected with the indicated plasmids individually and then resolved on an agarose gel. Transcripts of different types of expressed RNAs (indicated by white arrows) were probed with Dig-labeled antisense SNORD116-13 and their predicted sizes are labeled at the bottom of each NB. tRNA lys was used as markers for nuclear/cytoplasmic RNA isolation. Equal amounts of total, cytoplasmic and nuclear RNAs were loaded onto an agarose gel and rRNAs were used as the loading control. ( B ) Different types of RNAs, including intron sequences, a lncRNA and mRNAs, expressed from pEGFP-C1 are found in both the nucleus and in the cytoplasm. Total RNAs and fractionated nuclear and cytoplasmic RNAs were collected from HeLa cells transfected with the indicated plasmids individually and then resolved on an agarose gel. Transcripts of different types of expressed RNAs (indicated by white arrows) were probed with Dig-labeled egfp and their predicted sizes were labeled in the bottom of each NB. See (A) for details. ( C) Different types of RNAs expressed from pZW1-snoVector are retained in the nucleus, while those expressed from pEGFP-C1 are found in both the nucleus and the cytoplasm. ( D ) Different types of RNAs expressed from pcDNA3.0 vector are found in both the nucleus and the cytoplasm. In (A) and (B), white arrows indicate the expected RNAs expressed from different vectors. Assays were repeated and the same results were obtained. In (C) and (D), nuclear and cytoplasmic RNAs extracted from equal numbers of transfected HeLa cells under each indicated condition were assayed by RT-qPCR. The cytoplasmic distributed gapdh and the nuclear retained endogenous hNEAT1 were used as markers to indicate a qualified cytoplasmic and nuclear fractionation under each transfection condition. Error bars were calculated from three replicates. Also see supplemental information Figures S5–S6.

    Techniques Used: Transfection, Agarose Gel Electrophoresis, Labeling, Isolation, Plasmid Preparation, Quantitative RT-PCR, Fractionation

    mNEAT1 and its fragments expressed from snoVectors largely retain their endogenous characteristics in the nucleus. ( A ) The subcellular localization of mNEAT1 RNA from a pZW1-snoVector or a pEGFP-C1 vector. Left, HeLa cells were co-transfected with Flag-p54 nrb and pZW1-snoVector- mNEAT1 -FL or Flag-p54 nrb and pEGFP-C1- mNEAT1 -FL, followed by co-staining of Flag-p54 nrb (red) and mNEAT1 RNA (green). Representative images of all observed subcellular localization patterns of mNEAT1 expressed from these two vectors are shown (a-e). All nuclei were counterstained with DAPI. Scale bars 5 μm in all panels. Right, Quantitative analysis of each observed subcellular localization pattern of snoRNA-ended mNEAT1 or egfp - mNEAT1 counted from randomly selected microscopic fields. Note that the majority of expressed snoRNA-ended mNEAT1 RNA co-localizes with Flag-p54 nrb . ( B-E ) The subcellular localization of the expressed fragments of mNEAT1 RNA from either pZW1-snoVectors or pEGFP-C1 vectors. Representative images of all observed subcellular localization patterns (cytoplasmic localization; co-localization with p54 nrb in the nucleus; nonco-localization with p54 nrb in the nucleus) of expressed mNEAT1 fragments from these two vectors are shown. See (A) for details. Note that fragments of mNEAT1 expressed from snoVectors are predominately retained in the nucleus and co-localize with Flag-p54 nrb . ( F ) The majority of the snoVector expressed mNEAT1 RNAs do not localize to Cajal bodies or nucleoli. Left, HeLa cells were transfected with pZW1-snoVector- mNEAT1 -FL, followed by co-staining of Coilin (red) or Nucleolin (red) with mNEAT1 RNA (green). Representative images of all observed subcellular localization patterns (partial co-localization or nonco-localization) of snoRNA-ended mNEAT1 with Colin or Nucleolin are shown (a-d). Right, Quantitative analysis of each observed subcellular co-localization pattern of snoRNA-ended mNEAT1 with Colin or Nucleolin counted from randomly selected microscopic fields. In all panels, assays were repeated and the same results were obtained. Also see supplemental information Figures S3 and S4.
    Figure Legend Snippet: mNEAT1 and its fragments expressed from snoVectors largely retain their endogenous characteristics in the nucleus. ( A ) The subcellular localization of mNEAT1 RNA from a pZW1-snoVector or a pEGFP-C1 vector. Left, HeLa cells were co-transfected with Flag-p54 nrb and pZW1-snoVector- mNEAT1 -FL or Flag-p54 nrb and pEGFP-C1- mNEAT1 -FL, followed by co-staining of Flag-p54 nrb (red) and mNEAT1 RNA (green). Representative images of all observed subcellular localization patterns of mNEAT1 expressed from these two vectors are shown (a-e). All nuclei were counterstained with DAPI. Scale bars 5 μm in all panels. Right, Quantitative analysis of each observed subcellular localization pattern of snoRNA-ended mNEAT1 or egfp - mNEAT1 counted from randomly selected microscopic fields. Note that the majority of expressed snoRNA-ended mNEAT1 RNA co-localizes with Flag-p54 nrb . ( B-E ) The subcellular localization of the expressed fragments of mNEAT1 RNA from either pZW1-snoVectors or pEGFP-C1 vectors. Representative images of all observed subcellular localization patterns (cytoplasmic localization; co-localization with p54 nrb in the nucleus; nonco-localization with p54 nrb in the nucleus) of expressed mNEAT1 fragments from these two vectors are shown. See (A) for details. Note that fragments of mNEAT1 expressed from snoVectors are predominately retained in the nucleus and co-localize with Flag-p54 nrb . ( F ) The majority of the snoVector expressed mNEAT1 RNAs do not localize to Cajal bodies or nucleoli. Left, HeLa cells were transfected with pZW1-snoVector- mNEAT1 -FL, followed by co-staining of Coilin (red) or Nucleolin (red) with mNEAT1 RNA (green). Representative images of all observed subcellular localization patterns (partial co-localization or nonco-localization) of snoRNA-ended mNEAT1 with Colin or Nucleolin are shown (a-d). Right, Quantitative analysis of each observed subcellular co-localization pattern of snoRNA-ended mNEAT1 with Colin or Nucleolin counted from randomly selected microscopic fields. In all panels, assays were repeated and the same results were obtained. Also see supplemental information Figures S3 and S4.

    Techniques Used: Plasmid Preparation, Transfection, Staining

    snoVectors express RNAs in the nucleus. ( A ) A schematic drawing to show full-length (FL) mNEAT1 RNA and different fragments of it ( 9 ) inserted into either the sno-lncRNA region in pZW1-snoVector (top) or into the UTR region of pEGFP-C1, which was engineered with a stop codon immediately downstream of the EGFP ORF (Bottom). ( B ) mNEAT1 RNA and different fragments of it expressed from pZW1-snoVector are retained in the nucleus. Top, HeLa cells were transfected with indicated plasmids individually and fluorescence pictures were taken 36 h after transfection. Bottom, total RNAs and fractionated nuclear and cytoplasmic RNAs were collected from the same batch of transfected HeLa cells as shown above, and then resolved on an agarose gel. Transcripts of mNEAT1 RNA and different fragments of it were probed with a Dig-labeled antisense SNORD116-13; tRNA lys was used as marker for nuclear/cytoplasmic RNA isolation. Equal amounts of total, cytoplasmic and nuclear RNAs were loaded onto an agarose gel and rRNAs were used as the loading control. T, total RNAs; C, cytoplasmic RNAs; N, nuclear RNAs. ( C ) mNEAT1 RNA and different fragments of it expressed from pEGFP-C1 are at least partially exported to the cytoplasm. Top, HeLa cells were transfected individually with the indicated plasmids. Total RNAs and fractionated nuclear and cytoplasmic RNAs were collected 36 h after transfection, and then resolved on an agarose gel. Transcripts of mNEAT1 RNA and different fragments of it were probed with Dig-labeled egfp . See (B) for details. ( D ) RNAs expressed from pZW1-snoVector are absolutely retained in the nucleus, while those from pEGFP-C1 are not. Top, HeLa cells were transfected with each indicated plasmid in a pZW1-snoVector for 36 h. RNA in situ hybridizations were performed with Dig-labeled probes for mNEAT1 RNA and different fragments of it. Bottom, HeLa cells were transfected with each indicated plasmid in a pEGFP-C1 vector for 36 h. RNA in situ hybridizations were performed with Dig-labeled egfp . Representative images are shown for each transfection. White arrow heads represent the cytoplasmic signals of egfp - mNEAT1 -FL in cytoplasm in transfected HeLa cells. All nuclei were counterstained with DAPI. Scale bars 10 μm. ( E ) Subcellular distribution of transfected RNAs from different expression vectors. Quantitative analysis of the data from experiments shown in (D) is presented. More than 200 transfected cells were recorded randomly by confocal microscopy following each different transfection, and the percentage of each distinct nuclear/cytoplasmic localization pattern of RNAs transcribed from pZW1-snoVector or pEGFP-C1 was recorded. Note that mNEAT1 RNA and different fragments of it expressed from pZW1-snoVector are retained in the nucleus, while those from pEGFP-C1 are not. ( F ) mNEAT1 RNA and different fragments of it expressed from pZW1-snoVector are predominately retained in the nucleus. Nuclear and cytoplasmic RNAs extracted from equal numbers of transfected HeLa cells under each indicated condition were assayed by RT-qPCR. The cytoplasmic distributed gapdh and the nuclear retained endogenous hNEAT1 were used as markers to indicate a qualified cytoplasmic and nuclear fractionation under each transfection condition. Error bars were calculated from three replicates. In (B), (C) and (D), assays were repeated and the same results were obtained. Also see supplemental information Figure S2.
    Figure Legend Snippet: snoVectors express RNAs in the nucleus. ( A ) A schematic drawing to show full-length (FL) mNEAT1 RNA and different fragments of it ( 9 ) inserted into either the sno-lncRNA region in pZW1-snoVector (top) or into the UTR region of pEGFP-C1, which was engineered with a stop codon immediately downstream of the EGFP ORF (Bottom). ( B ) mNEAT1 RNA and different fragments of it expressed from pZW1-snoVector are retained in the nucleus. Top, HeLa cells were transfected with indicated plasmids individually and fluorescence pictures were taken 36 h after transfection. Bottom, total RNAs and fractionated nuclear and cytoplasmic RNAs were collected from the same batch of transfected HeLa cells as shown above, and then resolved on an agarose gel. Transcripts of mNEAT1 RNA and different fragments of it were probed with a Dig-labeled antisense SNORD116-13; tRNA lys was used as marker for nuclear/cytoplasmic RNA isolation. Equal amounts of total, cytoplasmic and nuclear RNAs were loaded onto an agarose gel and rRNAs were used as the loading control. T, total RNAs; C, cytoplasmic RNAs; N, nuclear RNAs. ( C ) mNEAT1 RNA and different fragments of it expressed from pEGFP-C1 are at least partially exported to the cytoplasm. Top, HeLa cells were transfected individually with the indicated plasmids. Total RNAs and fractionated nuclear and cytoplasmic RNAs were collected 36 h after transfection, and then resolved on an agarose gel. Transcripts of mNEAT1 RNA and different fragments of it were probed with Dig-labeled egfp . See (B) for details. ( D ) RNAs expressed from pZW1-snoVector are absolutely retained in the nucleus, while those from pEGFP-C1 are not. Top, HeLa cells were transfected with each indicated plasmid in a pZW1-snoVector for 36 h. RNA in situ hybridizations were performed with Dig-labeled probes for mNEAT1 RNA and different fragments of it. Bottom, HeLa cells were transfected with each indicated plasmid in a pEGFP-C1 vector for 36 h. RNA in situ hybridizations were performed with Dig-labeled egfp . Representative images are shown for each transfection. White arrow heads represent the cytoplasmic signals of egfp - mNEAT1 -FL in cytoplasm in transfected HeLa cells. All nuclei were counterstained with DAPI. Scale bars 10 μm. ( E ) Subcellular distribution of transfected RNAs from different expression vectors. Quantitative analysis of the data from experiments shown in (D) is presented. More than 200 transfected cells were recorded randomly by confocal microscopy following each different transfection, and the percentage of each distinct nuclear/cytoplasmic localization pattern of RNAs transcribed from pZW1-snoVector or pEGFP-C1 was recorded. Note that mNEAT1 RNA and different fragments of it expressed from pZW1-snoVector are retained in the nucleus, while those from pEGFP-C1 are not. ( F ) mNEAT1 RNA and different fragments of it expressed from pZW1-snoVector are predominately retained in the nucleus. Nuclear and cytoplasmic RNAs extracted from equal numbers of transfected HeLa cells under each indicated condition were assayed by RT-qPCR. The cytoplasmic distributed gapdh and the nuclear retained endogenous hNEAT1 were used as markers to indicate a qualified cytoplasmic and nuclear fractionation under each transfection condition. Error bars were calculated from three replicates. In (B), (C) and (D), assays were repeated and the same results were obtained. Also see supplemental information Figure S2.

    Techniques Used: Transfection, Fluorescence, Agarose Gel Electrophoresis, Labeling, Marker, Isolation, Plasmid Preparation, In Situ, Expressing, Confocal Microscopy, Quantitative RT-PCR, Fractionation

    28) Product Images from "GENE THERAPY OF CARCINOMA USING ULTRASOUND-TARGETED MICROBUBBLE DESTRUCTION"

    Article Title: GENE THERAPY OF CARCINOMA USING ULTRASOUND-TARGETED MICROBUBBLE DESTRUCTION

    Journal: Ultrasound in medicine & biology

    doi: 10.1016/j.ultrasmedbio.2010.11.011

    Postmortem histology of tumors after intravenous delivery of either pCMV-TK (a, c, d) or pEGFP-C1 (b)-loaded microbubbles and treatment with ultrasound and GCV. (a, b) Acellular zones ( arrows ) visible under H E stain. (c) Merged image of two sister
    Figure Legend Snippet: Postmortem histology of tumors after intravenous delivery of either pCMV-TK (a, c, d) or pEGFP-C1 (b)-loaded microbubbles and treatment with ultrasound and GCV. (a, b) Acellular zones ( arrows ) visible under H E stain. (c) Merged image of two sister

    Techniques Used: Staining

    Immunofluorescent staining of GFP in murine tumors 3 d after intravenous delivery of pEGFP-C1 bound to microbubbles and treatment with ultrasound (a, b) or no ultrasound (c). GFP-positive staining was seen both in hollow structures (a) as well as individual
    Figure Legend Snippet: Immunofluorescent staining of GFP in murine tumors 3 d after intravenous delivery of pEGFP-C1 bound to microbubbles and treatment with ultrasound (a, b) or no ultrasound (c). GFP-positive staining was seen both in hollow structures (a) as well as individual

    Techniques Used: Staining

    Postmortem TUNEL assays from murine tumors after intravenous injection of either pCMV-TK (a) or pEGFP-C1 (b)-loaded microbubbles and treatment with ultrasound and GCV.
    Figure Legend Snippet: Postmortem TUNEL assays from murine tumors after intravenous injection of either pCMV-TK (a) or pEGFP-C1 (b)-loaded microbubbles and treatment with ultrasound and GCV.

    Techniques Used: TUNEL Assay, Injection

    Growth of murine tumors after intravenous injection of either pCMV-TK (□) or pEGFP-C1 (▲)-loaded microbubbles and treated with ultrasound. Daily GCV injections began on day 3. Best-fit lines were calculated from all data points in each
    Figure Legend Snippet: Growth of murine tumors after intravenous injection of either pCMV-TK (□) or pEGFP-C1 (▲)-loaded microbubbles and treated with ultrasound. Daily GCV injections began on day 3. Best-fit lines were calculated from all data points in each

    Techniques Used: Injection

    29) Product Images from "Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids"

    Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043283

    Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
    Figure Legend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

    Techniques Used: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

    Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.
    Figure Legend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

    Techniques Used: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

    30) Product Images from "Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids"

    Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043283

    Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
    Figure Legend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

    Techniques Used: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

    Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.
    Figure Legend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

    Techniques Used: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

    31) Product Images from "Nuclear Accumulation of IE62, the Varicella-Zoster Virus (VZV) Major Transcriptional Regulatory Protein, Is Inhibited by Phosphorylation Mediated by the VZV Open Reading Frame 66 Protein Kinase"

    Article Title: Nuclear Accumulation of IE62, the Varicella-Zoster Virus (VZV) Major Transcriptional Regulatory Protein, Is Inhibited by Phosphorylation Mediated by the VZV Open Reading Frame 66 Protein Kinase

    Journal: Journal of Virology

    doi:

    Immunofluorescent images showing the nuclear localization of chimeric proteins and modulation by ORF66 protein. Protein was expressed from pKCMV62/61NL (A and B) or pKCMV62-EGFP (D and E) in the absence (A and D) or presence (B and E) of a threefold excess of HA epitope-tagged ORF66 protein. (C) HA-tagged ORF66 protein in the cells shown in panel B; (F) cellular location of the EGFP expressed from the commercial vector pEGFP-C1.
    Figure Legend Snippet: Immunofluorescent images showing the nuclear localization of chimeric proteins and modulation by ORF66 protein. Protein was expressed from pKCMV62/61NL (A and B) or pKCMV62-EGFP (D and E) in the absence (A and D) or presence (B and E) of a threefold excess of HA epitope-tagged ORF66 protein. (C) HA-tagged ORF66 protein in the cells shown in panel B; (F) cellular location of the EGFP expressed from the commercial vector pEGFP-C1.

    Techniques Used: Plasmid Preparation

    32) Product Images from "RNF8 mediates NONO degradation following UV-induced DNA damage to properly terminate ATR-CHK1 checkpoint signaling"

    Article Title: RNF8 mediates NONO degradation following UV-induced DNA damage to properly terminate ATR-CHK1 checkpoint signaling

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1166

    Mapping the region of NONO which confers instability in response to UV-induced DNA damage. ( A ) Schematic representation of NONO structure (RRM1 2: RNA recognition motifs 1 and 2, NOPS: NonA/paraspeckle domain, and CC: Coiled-coil domain) and strategy for mutagenesis. ( B ) Expression analysis of NONO deletion mutants. HEK-293 cells were transfected with pEGFP-C1-NONO and a series of pEF/myc/nuc/GFP-NONO mutant constructs. Twenty hours after transfection, cells were harvested and processed for Western blotting. ( C ) Analysis of protein stability for NONO deletion mutants after UV. HeLa cells were transfected with plasmids encoding GFP-tagged NONO and GFP-NLS-tagged NONO deletion mutants, and treated with UV (30 J/m 2 ) for the indicated time points. ( D ) Graphical representation of ( C ) illustrates GFP-tagged NONO and GFP-NLS-tagged NONO deletion mutants’ protein stability after UV. Error bars represent standard deviations from three independent experiments. ( E ) Generation of stable NONO deletion mutant based on the protein degradation information summarized in (C and D). Amino acid residues from 277 to 308 were deleted to generate a stable NONO deletion mutant. ( F ) Stable NONO deletion mutant localizes to the chromatin-enriched fraction. HeLa cells expressing FLAG-NONO and FLAG-NONO Δ277-308 were assayed for chromatin fractionation, and separated into soluble and chromatin-enriched fractions. The collected fractions were analyzed by Western blotting using an anti-H2AX antibody as a marker for the chromatin-enriched fraction and an anti-HSP90 antibody as a marker for the soluble fraction.
    Figure Legend Snippet: Mapping the region of NONO which confers instability in response to UV-induced DNA damage. ( A ) Schematic representation of NONO structure (RRM1 2: RNA recognition motifs 1 and 2, NOPS: NonA/paraspeckle domain, and CC: Coiled-coil domain) and strategy for mutagenesis. ( B ) Expression analysis of NONO deletion mutants. HEK-293 cells were transfected with pEGFP-C1-NONO and a series of pEF/myc/nuc/GFP-NONO mutant constructs. Twenty hours after transfection, cells were harvested and processed for Western blotting. ( C ) Analysis of protein stability for NONO deletion mutants after UV. HeLa cells were transfected with plasmids encoding GFP-tagged NONO and GFP-NLS-tagged NONO deletion mutants, and treated with UV (30 J/m 2 ) for the indicated time points. ( D ) Graphical representation of ( C ) illustrates GFP-tagged NONO and GFP-NLS-tagged NONO deletion mutants’ protein stability after UV. Error bars represent standard deviations from three independent experiments. ( E ) Generation of stable NONO deletion mutant based on the protein degradation information summarized in (C and D). Amino acid residues from 277 to 308 were deleted to generate a stable NONO deletion mutant. ( F ) Stable NONO deletion mutant localizes to the chromatin-enriched fraction. HeLa cells expressing FLAG-NONO and FLAG-NONO Δ277-308 were assayed for chromatin fractionation, and separated into soluble and chromatin-enriched fractions. The collected fractions were analyzed by Western blotting using an anti-H2AX antibody as a marker for the chromatin-enriched fraction and an anti-HSP90 antibody as a marker for the soluble fraction.

    Techniques Used: Mutagenesis, Expressing, Transfection, Construct, Western Blot, Fractionation, Marker

    33) Product Images from "Overexpression of miR-484 and miR-744 in Vero cells alters Dengue virus replication"

    Article Title: Overexpression of miR-484 and miR-744 in Vero cells alters Dengue virus replication

    Journal: Memórias do Instituto Oswaldo Cruz

    doi: 10.1590/0074-02760160404

    : miR-484 and miR-744 expression is downregulated in Vero cells expressing the Dengue virus (DENV) 3′ UTR. Vero cells were transfected with the pGUD1, pGUD2, or pGUD4 constructs or with the empty vector pEGFP-C1 and their effect on miRNA expression was determined. The expression of miR-484 (A) and miR-744 (B) was evaluated at 12, 24, 48, and 72 hpt by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and normalised to the untransfected control and to 18S RNA (2-ΔΔCt). Data from RT-qPCR are shown as median and bars are presented from three independent experiments. (*) Statistically significant difference compared to the control (p
    Figure Legend Snippet: : miR-484 and miR-744 expression is downregulated in Vero cells expressing the Dengue virus (DENV) 3′ UTR. Vero cells were transfected with the pGUD1, pGUD2, or pGUD4 constructs or with the empty vector pEGFP-C1 and their effect on miRNA expression was determined. The expression of miR-484 (A) and miR-744 (B) was evaluated at 12, 24, 48, and 72 hpt by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and normalised to the untransfected control and to 18S RNA (2-ΔΔCt). Data from RT-qPCR are shown as median and bars are presented from three independent experiments. (*) Statistically significant difference compared to the control (p

    Techniques Used: Expressing, Transfection, Construct, Plasmid Preparation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    : cellular miRNAs alter the expression of GFP-fused to the 3′ UTR of Dengue virus (DENV) RNA. (A) Vero cells were transfected with pGUD1, pGUD2, pGUD4, or pEGFP-C1 and the GFP expression was determined by western blotting after 24 h, using an anti-GFP antibody; (b) band intensities quantified by densitometry; (C) Vero cells were co-transfected with pGUD1, pGUD2, pGUD4, or pEGFP-C1 and pEZX-mR03-miR-484 or pEZX-mR03-miR-744, and GFP expression was then determined by western blot after 24 h using an anti-GFP antibody; (D) band intensities quantified by densitometry. Data are shown as Median and Range (two way ANOVA). Three independent replicates were performed for each experiment. (*) Statistically significant difference compared to the control (p
    Figure Legend Snippet: : cellular miRNAs alter the expression of GFP-fused to the 3′ UTR of Dengue virus (DENV) RNA. (A) Vero cells were transfected with pGUD1, pGUD2, pGUD4, or pEGFP-C1 and the GFP expression was determined by western blotting after 24 h, using an anti-GFP antibody; (b) band intensities quantified by densitometry; (C) Vero cells were co-transfected with pGUD1, pGUD2, pGUD4, or pEGFP-C1 and pEZX-mR03-miR-484 or pEZX-mR03-miR-744, and GFP expression was then determined by western blot after 24 h using an anti-GFP antibody; (D) band intensities quantified by densitometry. Data are shown as Median and Range (two way ANOVA). Three independent replicates were performed for each experiment. (*) Statistically significant difference compared to the control (p

    Techniques Used: Expressing, Transfection, Western Blot

    34) Product Images from "DNA prime and peptide boost immunization protocol encoding the Toxoplasma gondii GRA4 induces strong protective immunity in BALB/c mice"

    Article Title: DNA prime and peptide boost immunization protocol encoding the Toxoplasma gondii GRA4 induces strong protective immunity in BALB/c mice

    Journal: BMC Infectious Diseases

    doi: 10.1186/1471-2334-13-494

    Fluorescence microscopy images of HEK293T cells and western blotting analysis. (A) HEK293 cells transfected with empty vector pEGFP-C1. (B) HEK293 cells transfected with pGRA4. (C) untransfected HEK cells. (D) protein marker (lane M), pGRA4-transfected cells (lane 1), pEGFP-transfected cells (lane 2), untransfected cells (lane 3).
    Figure Legend Snippet: Fluorescence microscopy images of HEK293T cells and western blotting analysis. (A) HEK293 cells transfected with empty vector pEGFP-C1. (B) HEK293 cells transfected with pGRA4. (C) untransfected HEK cells. (D) protein marker (lane M), pGRA4-transfected cells (lane 1), pEGFP-transfected cells (lane 2), untransfected cells (lane 3).

    Techniques Used: Fluorescence, Microscopy, Western Blot, Transfection, Plasmid Preparation, Marker

    35) Product Images from "Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids"

    Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043283

    Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
    Figure Legend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

    Techniques Used: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

    Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.
    Figure Legend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

    Techniques Used: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

    Related Articles

    Quantitative RT-PCR:

    Article Title: Altered Patterns of Cellular Gene Expression in Dermal Microvascular Endothelial Cells Infected with Kaposi's Sarcoma-Associated Herpesvirus
    Article Snippet: Surprisingly, while confirming most of the gene array data, the real-time RT-PCR results nevertheless indicated that the +7.6 and +4.9 values for metallothionine H1 and CD14 in infected compared to uninfected RNA in the Clontech gene array with batch 1 RNA (and matching upregulation by RT-PCR in batch 3 and 4 RNA) were not reproducible in the Incyte batch 2 RNA preparation or in the Incyte array data for CD14. .. The real-time RT-PCR results for IL-8 (−50 and −35) also resolved the previously inconsistent array data, where only the Clontech +TPA value of −4.6 appears to be accurate, compared to the Clontech +TPA value of +2.1 and the not significantly regulated results for both of the Incyte surveys. .. Similarly, the real-time RT-PCR results for connective tissue growth factor with batch 2 RNA are far more consistent with the Incyte array result than with the Clontech array results.

    Amplification:

    Article Title: P311 induces a TGF-?1-independent, nonfibrogenic myofibroblast phenotype
    Article Snippet: The cells were cultured for either 1 or 18 hours, the first time point representing undifferentiated embryonic mesenchymal cells and the second representing cells undergoing SM differentiation. .. The mRNA from the two cultures was amplified using the SMART cDNA synthesis kit (CLONTECH Laboratories Inc., Palo Alto, California, USA), and PCR-Select (CLONTECH Laboratories Inc.) was then used for suppressive subtraction hybridization. ..

    Polymerase Chain Reaction:

    Article Title: P311 induces a TGF-?1-independent, nonfibrogenic myofibroblast phenotype
    Article Snippet: The cells were cultured for either 1 or 18 hours, the first time point representing undifferentiated embryonic mesenchymal cells and the second representing cells undergoing SM differentiation. .. The mRNA from the two cultures was amplified using the SMART cDNA synthesis kit (CLONTECH Laboratories Inc., Palo Alto, California, USA), and PCR-Select (CLONTECH Laboratories Inc.) was then used for suppressive subtraction hybridization. ..

    Article Title: Initiation of liver growth by tumor necrosis factor: Deficient liver regeneration in mice lacking type I tumor necrosis factor receptor
    Article Snippet: Amplified products obtained with 30 cycles were electrophoresed in 2% agarose gels and stained with ethidium bromide. .. Quantitation of IL-6 mRNA was done by competitive PCR using the PCR Mimic Protocol (CLONTECH). ..

    Article Title: Hepatic Insulin Resistance Directly Promotes Formation of Cholesterol Gallstones
    Article Snippet: Quantitative real-time PCR analysis of FOXO1 transgenic livers was performed as described previously and expression levels normalized to L32 ribosomal protein. .. In all other experiments, RNA (RNeasy, Qiagen) was isolated from mouse liver and used to direct cDNA synthesis (RT for PCR, Clontech). .. Quantitative real-time PCR was performed in a fluorescent temperature cycler (ABI) with either SYBR Green Master Mix (Roche, Mannheim, Germany) or the Taqman Universal master mix (ABI; primers purchased through Assay on Demand) .

    Article Title: BMPER, a Novel Endothelial Cell Precursor-Derived Protein, Antagonizes Bone Morphogenetic Protein Signaling and Endothelial Cell Differentiation
    Article Snippet: The cDNA libraries were amplified by PCR with the internal priming site. .. An flk-1-enriched cDNA library was created by subtracting the flk-1-negative library from the flk-1-positive library by using subtractive PCR technology (PCR-SELECT; Clontech). .. The resulting cDNA library was cloned into vector pCR2.1 (Invitrogen), transformed into Escherichia coli , plated on Luria-Bertani-ampicillin agar, and screened for inserts with blue-white color selection.

    Hybridization:

    Article Title: P311 induces a TGF-?1-independent, nonfibrogenic myofibroblast phenotype
    Article Snippet: The cells were cultured for either 1 or 18 hours, the first time point representing undifferentiated embryonic mesenchymal cells and the second representing cells undergoing SM differentiation. .. The mRNA from the two cultures was amplified using the SMART cDNA synthesis kit (CLONTECH Laboratories Inc., Palo Alto, California, USA), and PCR-Select (CLONTECH Laboratories Inc.) was then used for suppressive subtraction hybridization. ..

    Quantitation Assay:

    Article Title: Initiation of liver growth by tumor necrosis factor: Deficient liver regeneration in mice lacking type I tumor necrosis factor receptor
    Article Snippet: Amplified products obtained with 30 cycles were electrophoresed in 2% agarose gels and stained with ethidium bromide. .. Quantitation of IL-6 mRNA was done by competitive PCR using the PCR Mimic Protocol (CLONTECH). ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Identification of two novel CT antigens and their capacity to elicit antibody response in hepatocellular carcinoma patients
    Article Snippet: Consequently, Unigene and SAGE can be used to analyse the spatial and temporal expression pattern of a given gene for predicting the functional information of the gene. .. RT–PCR The mRNA expression pattern was assayed by RT–PCR using panels of commercially available cDNA (Clontech, Palo Alto, CA, USA). ..

    Expressing:

    Article Title: Identification of two novel CT antigens and their capacity to elicit antibody response in hepatocellular carcinoma patients
    Article Snippet: Consequently, Unigene and SAGE can be used to analyse the spatial and temporal expression pattern of a given gene for predicting the functional information of the gene. .. RT–PCR The mRNA expression pattern was assayed by RT–PCR using panels of commercially available cDNA (Clontech, Palo Alto, CA, USA). ..

    Isolation:

    Article Title: Hepatic Insulin Resistance Directly Promotes Formation of Cholesterol Gallstones
    Article Snippet: Quantitative real-time PCR analysis of FOXO1 transgenic livers was performed as described previously and expression levels normalized to L32 ribosomal protein. .. In all other experiments, RNA (RNeasy, Qiagen) was isolated from mouse liver and used to direct cDNA synthesis (RT for PCR, Clontech). .. Quantitative real-time PCR was performed in a fluorescent temperature cycler (ABI) with either SYBR Green Master Mix (Roche, Mannheim, Germany) or the Taqman Universal master mix (ABI; primers purchased through Assay on Demand) .

    cDNA Library Assay:

    Article Title: BMPER, a Novel Endothelial Cell Precursor-Derived Protein, Antagonizes Bone Morphogenetic Protein Signaling and Endothelial Cell Differentiation
    Article Snippet: The cDNA libraries were amplified by PCR with the internal priming site. .. An flk-1-enriched cDNA library was created by subtracting the flk-1-negative library from the flk-1-positive library by using subtractive PCR technology (PCR-SELECT; Clontech). .. The resulting cDNA library was cloned into vector pCR2.1 (Invitrogen), transformed into Escherichia coli , plated on Luria-Bertani-ampicillin agar, and screened for inserts with blue-white color selection.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    TaKaRa pegfp c1
    Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected <t>pEGFP-C1</t> (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
    Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp c1/product/TaKaRa
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pegfp c1 - by Bioz Stars, 2021-06
    86/100 stars
      Buy from Supplier

    Image Search Results


    Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

    Journal: PLoS ONE

    Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

    doi: 10.1371/journal.pone.0043283

    Figure Lengend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

    Article Snippet: In the case of pRFP-T, exchange of resistance completely abolished the inhibitory effect while there was a residual inhibition observed in the case of pEGFP-C1.

    Techniques: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

    Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

    Journal: PLoS ONE

    Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

    doi: 10.1371/journal.pone.0043283

    Figure Lengend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

    Article Snippet: In the case of pRFP-T, exchange of resistance completely abolished the inhibitory effect while there was a residual inhibition observed in the case of pEGFP-C1.

    Techniques: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

    NSAP1 augments HCV IRES-dependent translation. (A) Schematic diagrams of dicistronic reporter plasmids used for monitoring efficiency of cap- and IRES-dependent translation in vivo. The vectors contained the cytomegalovirus (CMV) immediate early enhancer-promoter (CMV pro.) to direct transcription in vivo. The transcription start site in the CMV promoter is indicated by open arrows. (B) (i) Translation activities of IRES-containing dicistronic mRNAs in 293T cells. Dicistronic reporter constructs (0.5 μg each of pRH402F [lanes 1 to 4], pRPF [lanes 5 to 8], pREF [lanes 9 to 12], and pRM531F [lanes 13 to 16]) and variable amounts of effecter pEGFP-c1/NSAP1 (2.5 μg [lanes 2, 6, 10, and 14], 5 μg [lanes 3, 7, 11, and 15], and 10 μg [lanes 4, 8, 12, and 16]) and negative control effecter pEGFP-c1 (10 μg [lanes 1, 5, 9, and 13], 7.5 μg [lanes 2, 6, 10, and 14], and 5 μg [lanes 3, 7, 11, and 15]) were cotransfected with the control plasmid pCMV · SPORT-βgal (Invitrogen) to monitor the effect of NSAP1 on IRES activities. Forty-eight hours after transfection by electroporation, the cells were harvested and their luciferase activities were measured. For IRES-dependent translation, firefly luciferase activity, which was directed by pRH402F, was arbitrarily set to 1. For cap-dependent translation, Renilla luciferase activity, which was directed by pRH402F, was arbitrarily set to 1. The transfection efficiency was also determined for each luciferase value relative to that of the β-galactosidase activity of the transfection control. Error bars represent standard deviation values. (ii) Expression profiles of GFP-NSAP1. Immunoblot analysis was performed with antibodies against GFP, NSAP1, and actin.

    Journal: Molecular and Cellular Biology

    Article Title: A Cellular RNA-Binding Protein Enhances Internal Ribosomal Entry Site-Dependent Translation through an Interaction Downstream of the Hepatitis C Virus Polyprotein Initiation Codon

    doi: 10.1128/MCB.24.18.7878-7890.2004

    Figure Lengend Snippet: NSAP1 augments HCV IRES-dependent translation. (A) Schematic diagrams of dicistronic reporter plasmids used for monitoring efficiency of cap- and IRES-dependent translation in vivo. The vectors contained the cytomegalovirus (CMV) immediate early enhancer-promoter (CMV pro.) to direct transcription in vivo. The transcription start site in the CMV promoter is indicated by open arrows. (B) (i) Translation activities of IRES-containing dicistronic mRNAs in 293T cells. Dicistronic reporter constructs (0.5 μg each of pRH402F [lanes 1 to 4], pRPF [lanes 5 to 8], pREF [lanes 9 to 12], and pRM531F [lanes 13 to 16]) and variable amounts of effecter pEGFP-c1/NSAP1 (2.5 μg [lanes 2, 6, 10, and 14], 5 μg [lanes 3, 7, 11, and 15], and 10 μg [lanes 4, 8, 12, and 16]) and negative control effecter pEGFP-c1 (10 μg [lanes 1, 5, 9, and 13], 7.5 μg [lanes 2, 6, 10, and 14], and 5 μg [lanes 3, 7, 11, and 15]) were cotransfected with the control plasmid pCMV · SPORT-βgal (Invitrogen) to monitor the effect of NSAP1 on IRES activities. Forty-eight hours after transfection by electroporation, the cells were harvested and their luciferase activities were measured. For IRES-dependent translation, firefly luciferase activity, which was directed by pRH402F, was arbitrarily set to 1. For cap-dependent translation, Renilla luciferase activity, which was directed by pRH402F, was arbitrarily set to 1. The transfection efficiency was also determined for each luciferase value relative to that of the β-galactosidase activity of the transfection control. Error bars represent standard deviation values. (ii) Expression profiles of GFP-NSAP1. Immunoblot analysis was performed with antibodies against GFP, NSAP1, and actin.

    Article Snippet: For the construction of pEGFP-c1/NSAP1, pSK(−)/NSAP1 was treated with EcoRI-Klenow-Asp718, and the DNA fragment was cloned into pEGFP-c1 (Clontech) treated with NheI-Klenow-Asp718.

    Techniques: In Vivo, Construct, Negative Control, Plasmid Preparation, Transfection, Electroporation, Luciferase, Activity Assay, Standard Deviation, Expressing

    Association of FRL and truncated FRL proteins with Rac1 in vitro and in vivo. (A) Precipitation of FRL by the GTPγS- or GDP-bound form of Rac1. P388D1 cell lysates were incubated with GST-RhoA, GST-Rac1, GST-Cdc42Hs, or GST, each of which was preloaded with either GTPγs or GDP. GST fusion proteins were precipitated by glutathione-Sepharose 4B beads, and the pellets were analyzed by immunoblotting with an anti-FRL antibody. (B) In vivo association of FRL with Rac1. P388D1 cell lysates were immobilized on protein A-agarose beads without (lane 1) or with (lane 2) anti-Rac1. The precipitates were analyzed by Western blotting with an anti-FRL antibody and an anti-Rac1 antibody. (C) Structure of truncated FRLs. FH3-FRL contains only the FH3 domain, FH1+FH3-FRL contains the FH1 and FH3 domains, and FH2-FRL contains only the FH2 domain. N1 consists of the N-terminal 80 amino acids, and N2 consists of the N-terminal 216 amino acids. (D) FRL associates with Rac1 at its N-terminal region containing the FH3 domain. COS7 cells transfected with each expression vector (FH3-pEGFP-C1, FH1+FH3-pEGFP-C1, FH2-pEGFP-C1, N1-pEGFP-C1, or N2-pEGFP-C1) were lysed, incubated with GST-Rac1, and precipitated by glutathione-Sepharose 4B. The pellets were analyzed by immunoblotting with an anti-GFP antibody.

    Journal: Molecular and Cellular Biology

    Article Title: FRL, a Novel Formin-Related Protein, Binds to Rac and Regulates Cell Motility and Survival of Macrophages

    doi:

    Figure Lengend Snippet: Association of FRL and truncated FRL proteins with Rac1 in vitro and in vivo. (A) Precipitation of FRL by the GTPγS- or GDP-bound form of Rac1. P388D1 cell lysates were incubated with GST-RhoA, GST-Rac1, GST-Cdc42Hs, or GST, each of which was preloaded with either GTPγs or GDP. GST fusion proteins were precipitated by glutathione-Sepharose 4B beads, and the pellets were analyzed by immunoblotting with an anti-FRL antibody. (B) In vivo association of FRL with Rac1. P388D1 cell lysates were immobilized on protein A-agarose beads without (lane 1) or with (lane 2) anti-Rac1. The precipitates were analyzed by Western blotting with an anti-FRL antibody and an anti-Rac1 antibody. (C) Structure of truncated FRLs. FH3-FRL contains only the FH3 domain, FH1+FH3-FRL contains the FH1 and FH3 domains, and FH2-FRL contains only the FH2 domain. N1 consists of the N-terminal 80 amino acids, and N2 consists of the N-terminal 216 amino acids. (D) FRL associates with Rac1 at its N-terminal region containing the FH3 domain. COS7 cells transfected with each expression vector (FH3-pEGFP-C1, FH1+FH3-pEGFP-C1, FH2-pEGFP-C1, N1-pEGFP-C1, or N2-pEGFP-C1) were lysed, incubated with GST-Rac1, and precipitated by glutathione-Sepharose 4B. The pellets were analyzed by immunoblotting with an anti-GFP antibody.

    Article Snippet: The plasmids of full-length frl α cDNA (FULL-pEGFP-C1) and truncated forms (FH3-pEGFP-C1, FH1+FH3-pEGFP-C1, FH2-pEGFP-C1, N1-pEGFP-C1, and N2-pEGFP-C1) (see Fig. C) were also inserted into the C-terminal protein fusion vector pEGFP-C1 (Clontech).

    Techniques: In Vitro, In Vivo, Incubation, Western Blot, Transfection, Expressing, Plasmid Preparation

    FRL associates with profilin. (A) Interaction of FRL and profilin in vitro. P388D1 cell lysates were incubated with GST-profilin I or with GST-profilin II and then precipitated by glutathione-Sepharose 4B beads, and the pellets were analyzed by immunoblotting with anti-FRL, anti-GST, and anti-actin antibodies. (B) In vivo association of FRL with profilin. P388D1 cell lysates were immobilized on protein A-agarose beads without (lane 1) or with (lane 2) an anti-FRL antibody. The precipitates were analyzed by Western blotting with anti-FRL and anti-profilin antibodies. (C) The FH1 domain of FRL binds to profilin. COS7 cells transfected with each expression vector (FH3-pEGFP-C1, FH1+FH3-pEGFP-C1, and FH2-pEGFP-C1) were lysed, incubated with GST-profilin I, and precipitated by glutathione-Sepharose 4B beads. The pellets were analyzed by immunoblotting with anti-GFP and anti-actin antibodies.

    Journal: Molecular and Cellular Biology

    Article Title: FRL, a Novel Formin-Related Protein, Binds to Rac and Regulates Cell Motility and Survival of Macrophages

    doi:

    Figure Lengend Snippet: FRL associates with profilin. (A) Interaction of FRL and profilin in vitro. P388D1 cell lysates were incubated with GST-profilin I or with GST-profilin II and then precipitated by glutathione-Sepharose 4B beads, and the pellets were analyzed by immunoblotting with anti-FRL, anti-GST, and anti-actin antibodies. (B) In vivo association of FRL with profilin. P388D1 cell lysates were immobilized on protein A-agarose beads without (lane 1) or with (lane 2) an anti-FRL antibody. The precipitates were analyzed by Western blotting with anti-FRL and anti-profilin antibodies. (C) The FH1 domain of FRL binds to profilin. COS7 cells transfected with each expression vector (FH3-pEGFP-C1, FH1+FH3-pEGFP-C1, and FH2-pEGFP-C1) were lysed, incubated with GST-profilin I, and precipitated by glutathione-Sepharose 4B beads. The pellets were analyzed by immunoblotting with anti-GFP and anti-actin antibodies.

    Article Snippet: The plasmids of full-length frl α cDNA (FULL-pEGFP-C1) and truncated forms (FH3-pEGFP-C1, FH1+FH3-pEGFP-C1, FH2-pEGFP-C1, N1-pEGFP-C1, and N2-pEGFP-C1) (see Fig. C) were also inserted into the C-terminal protein fusion vector pEGFP-C1 (Clontech).

    Techniques: In Vitro, Incubation, In Vivo, Western Blot, Transfection, Expressing, Plasmid Preparation

    Overexpression of FH1+FH3-FRL or FULL-FRL protein restored the cell adhesion, migration, and proliferation abilities of doxycycline-treated FH3/P388D1 cells. FH3/P388D1 cells were transfected with FH1+FH3-pEGFP-C1 (FH1+FH3), FULL-pEGFP-C1 (FULL), and pEGFP-C1 vector only (control), and then cultured with doxycycline for 24 h. (A) The cells expressing FH1+FH3-FRL or FULL-FRL together with FH3-FRL truncated protein spread and formed ruffles upon stimulation with SDF-1. In the panels on the left, cells were stained with rhodamine-conjugated phalloidin for actin staining. Right panels show GFP-positive cells. Note that the cell without expression of transfected FRL-GFP fusion protein does not show spreading or the formation of membrane ruffles. (B) Adhesion assay. Expression of FH1+FH3 or FULL-FRL also rescued the failure of FH3/P388D1 cells to adhere to fibronectin. (C) Migration of FH3/P388D1 against SDF-1 after expression of FH1+FH3-FRL or FULL-FRL protein together with FH3-FRL truncated protein. (D) [ 3 H]thymidine incorporation of FULL-FRL- or FH1+FH3-FRL-transfected FH3/P388D1 cells at 72 h after the addition of doxycycline (1.0 μg/ml). Proliferation of FH3/P388D1 cells was restored by expression of FH1+FH3-FRL or FULL-FRL protein, even in the presence of the truncated FH3-FRL induced by doxycycline.

    Journal: Molecular and Cellular Biology

    Article Title: FRL, a Novel Formin-Related Protein, Binds to Rac and Regulates Cell Motility and Survival of Macrophages

    doi:

    Figure Lengend Snippet: Overexpression of FH1+FH3-FRL or FULL-FRL protein restored the cell adhesion, migration, and proliferation abilities of doxycycline-treated FH3/P388D1 cells. FH3/P388D1 cells were transfected with FH1+FH3-pEGFP-C1 (FH1+FH3), FULL-pEGFP-C1 (FULL), and pEGFP-C1 vector only (control), and then cultured with doxycycline for 24 h. (A) The cells expressing FH1+FH3-FRL or FULL-FRL together with FH3-FRL truncated protein spread and formed ruffles upon stimulation with SDF-1. In the panels on the left, cells were stained with rhodamine-conjugated phalloidin for actin staining. Right panels show GFP-positive cells. Note that the cell without expression of transfected FRL-GFP fusion protein does not show spreading or the formation of membrane ruffles. (B) Adhesion assay. Expression of FH1+FH3 or FULL-FRL also rescued the failure of FH3/P388D1 cells to adhere to fibronectin. (C) Migration of FH3/P388D1 against SDF-1 after expression of FH1+FH3-FRL or FULL-FRL protein together with FH3-FRL truncated protein. (D) [ 3 H]thymidine incorporation of FULL-FRL- or FH1+FH3-FRL-transfected FH3/P388D1 cells at 72 h after the addition of doxycycline (1.0 μg/ml). Proliferation of FH3/P388D1 cells was restored by expression of FH1+FH3-FRL or FULL-FRL protein, even in the presence of the truncated FH3-FRL induced by doxycycline.

    Article Snippet: The plasmids of full-length frl α cDNA (FULL-pEGFP-C1) and truncated forms (FH3-pEGFP-C1, FH1+FH3-pEGFP-C1, FH2-pEGFP-C1, N1-pEGFP-C1, and N2-pEGFP-C1) (see Fig. C) were also inserted into the C-terminal protein fusion vector pEGFP-C1 (Clontech).

    Techniques: Over Expression, Migration, Transfection, Plasmid Preparation, Cell Culture, Expressing, Staining, Cell Adhesion Assay

    RT-PCR products of five recombinant plasmids. M: 100-6000 bp DNA Marker. (A) Lane 1-20 showed the RT-PCR results of BxPC-3 cells tranfected with five recombinant plasmids (Mu0, Mu1, Mu2, Mu3,Mu4) at 12 h, 24 h, 36 h, 48 h respectively, using the primer of β-actin. (B) Lane 1-20 showed the RT-PCR results of BxPC-3 cells tranfected with five recombinant plasmids (Mu0, Mu1, Mu2, Mu3, Mu4) at 12 h, 24 h, 36 h, 48 h respectively, using the primer of hIL-18 full length. Lane 21 is negative control. (C) Lane1-20 showed the RT-PCR results of BxPC-3 cells tranfected with five recombinant plasmids (Mu0, Mu1, Mu2, Mu3, Mu4) at 12 h, 24 h, 36 h, 48 h respectively, using the original primers. (D) Control. Lane 1-4 and lane 5-8 showed RT-PCR results of BxPC-3 cells tranfected with pEGFP-C1 and untreated BxPC-3 cells cultivated at 12 h, 24 h, 36 h, 48 h respectively, using the primer of hIL-18 full length. Lane 9-12 and lane 13-16 showed RT-PCR results of BxPC-3 cells with the same treatment (according to Lane 1-4 and 5-8) using the primer of β-actin.

    Journal: International Journal of Medical Sciences

    Article Title: Intracellular Distributing and Interferon-? Secretion of Human Interleukin-18 in BxPC-3 Cells

    doi: 10.7150/ijms.6875

    Figure Lengend Snippet: RT-PCR products of five recombinant plasmids. M: 100-6000 bp DNA Marker. (A) Lane 1-20 showed the RT-PCR results of BxPC-3 cells tranfected with five recombinant plasmids (Mu0, Mu1, Mu2, Mu3,Mu4) at 12 h, 24 h, 36 h, 48 h respectively, using the primer of β-actin. (B) Lane 1-20 showed the RT-PCR results of BxPC-3 cells tranfected with five recombinant plasmids (Mu0, Mu1, Mu2, Mu3, Mu4) at 12 h, 24 h, 36 h, 48 h respectively, using the primer of hIL-18 full length. Lane 21 is negative control. (C) Lane1-20 showed the RT-PCR results of BxPC-3 cells tranfected with five recombinant plasmids (Mu0, Mu1, Mu2, Mu3, Mu4) at 12 h, 24 h, 36 h, 48 h respectively, using the original primers. (D) Control. Lane 1-4 and lane 5-8 showed RT-PCR results of BxPC-3 cells tranfected with pEGFP-C1 and untreated BxPC-3 cells cultivated at 12 h, 24 h, 36 h, 48 h respectively, using the primer of hIL-18 full length. Lane 9-12 and lane 13-16 showed RT-PCR results of BxPC-3 cells with the same treatment (according to Lane 1-4 and 5-8) using the primer of β-actin.

    Article Snippet: 2.1 Plasmids and cell culture Eukaryotic expression vector pEGFP-C1 was purchased from Clontech (Palo Alto, USA). pGEM-T-hIL-18 was conserved in our laboratory.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Recombinant, Marker, Negative Control

    Localization of enhanced green fluorescent protein (EGFP)-tagged hIL-18 recombinant plasmids inBxPC-3 cells. Confocal micrographs of transfected BxPC-3 cells expressing EGFP-tagged hIL-18 recombinant plasmid at 12 h post-transfection (A) 24 h post-transfection, (B) 36 h post-transfection, (C) 48 h post-transfection, (D) and composite photo (E) respectively . Images were taken in the plane where maximum fluorescence appeared. Transfected cells presented uniform fluorescence throughout the cytoplasm and the nucleus 12 h post-transfection, and the fluorescence intensity of transfected cells with pEGFP-C1 are durable(A-D), fluorescence intensity of other cells are decreasing. But from 24 h and 36 h post-transfection, the fluorescence of the hIL-18 Mu1 and Mu2 appeared targeted to the membranous region of the BxPC-3 cells. Magnification, 200×.

    Journal: International Journal of Medical Sciences

    Article Title: Intracellular Distributing and Interferon-? Secretion of Human Interleukin-18 in BxPC-3 Cells

    doi: 10.7150/ijms.6875

    Figure Lengend Snippet: Localization of enhanced green fluorescent protein (EGFP)-tagged hIL-18 recombinant plasmids inBxPC-3 cells. Confocal micrographs of transfected BxPC-3 cells expressing EGFP-tagged hIL-18 recombinant plasmid at 12 h post-transfection (A) 24 h post-transfection, (B) 36 h post-transfection, (C) 48 h post-transfection, (D) and composite photo (E) respectively . Images were taken in the plane where maximum fluorescence appeared. Transfected cells presented uniform fluorescence throughout the cytoplasm and the nucleus 12 h post-transfection, and the fluorescence intensity of transfected cells with pEGFP-C1 are durable(A-D), fluorescence intensity of other cells are decreasing. But from 24 h and 36 h post-transfection, the fluorescence of the hIL-18 Mu1 and Mu2 appeared targeted to the membranous region of the BxPC-3 cells. Magnification, 200×.

    Article Snippet: 2.1 Plasmids and cell culture Eukaryotic expression vector pEGFP-C1 was purchased from Clontech (Palo Alto, USA). pGEM-T-hIL-18 was conserved in our laboratory.

    Techniques: Recombinant, Transfection, Expressing, Plasmid Preparation, Fluorescence