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  • 95
    TaKaRa pegfp c1 takara clontech
    Pegfp C1 Takara Clontech, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    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: 91/100, based on 888 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    egfp  (TaKaRa)
    92
    TaKaRa egfp
    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.
    Egfp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 7287 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    TaKaRa plasmid pegfp c1
    MC MV and MC MVΔD, but not MC MVΔF and BR MV, rescue melanosome distribution in dilute melanocytes. Shown is the distribution of melanosomes in brightfield (A, D, G, and J), the cell shape in phase contrast (B, E, H, and K), and the distribution of GFP fluorescence due primarily to unfused GFP expressed from plasmid <t>pEGFP</t> C1 (C, F, I, and L), which was coinjected to ensure accurate identification of all injected cells (see text), in dilute melanocytes microinjected with MC MV (A–C), MC MVΔD (D–F), MC MVΔF (G–I), or BR MV (J–L). Bars, 11.5 μm.
    Plasmid Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 719 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa bglii ecori digested pegfp c1
    MC MV and MC MVΔD, but not MC MVΔF and BR MV, rescue melanosome distribution in dilute melanocytes. Shown is the distribution of melanosomes in brightfield (A, D, G, and J), the cell shape in phase contrast (B, E, H, and K), and the distribution of GFP fluorescence due primarily to unfused GFP expressed from plasmid <t>pEGFP</t> C1 (C, F, I, and L), which was coinjected to ensure accurate identification of all injected cells (see text), in dilute melanocytes microinjected with MC MV (A–C), MC MVΔD (D–F), MC MVΔF (G–I), or BR MV (J–L). Bars, 11.5 μm.
    Bglii Ecori Digested Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa cdnas pegfp c1
    MC MV and MC MVΔD, but not MC MVΔF and BR MV, rescue melanosome distribution in dilute melanocytes. Shown is the distribution of melanosomes in brightfield (A, D, G, and J), the cell shape in phase contrast (B, E, H, and K), and the distribution of GFP fluorescence due primarily to unfused GFP expressed from plasmid <t>pEGFP</t> C1 (C, F, I, and L), which was coinjected to ensure accurate identification of all injected cells (see text), in dilute melanocytes microinjected with MC MV (A–C), MC MVΔD (D–F), MC MVΔF (G–I), or BR MV (J–L). Bars, 11.5 μm.
    Cdnas Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa complementary dnas pegfp c1
    MC MV and MC MVΔD, but not MC MVΔF and BR MV, rescue melanosome distribution in dilute melanocytes. Shown is the distribution of melanosomes in brightfield (A, D, G, and J), the cell shape in phase contrast (B, E, H, and K), and the distribution of GFP fluorescence due primarily to unfused GFP expressed from plasmid <t>pEGFP</t> C1 (C, F, I, and L), which was coinjected to ensure accurate identification of all injected cells (see text), in dilute melanocytes microinjected with MC MV (A–C), MC MVΔD (D–F), MC MVΔF (G–I), or BR MV (J–L). Bars, 11.5 μm.
    Complementary Dnas Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa transfections pegfp c1
    MC MV and MC MVΔD, but not MC MVΔF and BR MV, rescue melanosome distribution in dilute melanocytes. Shown is the distribution of melanosomes in brightfield (A, D, G, and J), the cell shape in phase contrast (B, E, H, and K), and the distribution of GFP fluorescence due primarily to unfused GFP expressed from plasmid <t>pEGFP</t> C1 (C, F, I, and L), which was coinjected to ensure accurate identification of all injected cells (see text), in dilute melanocytes microinjected with MC MV (A–C), MC MVΔD (D–F), MC MVΔF (G–I), or BR MV (J–L). Bars, 11.5 μm.
    Transfections Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa sali digested pegfp c1
    MC MV and MC MVΔD, but not MC MVΔF and BR MV, rescue melanosome distribution in dilute melanocytes. Shown is the distribution of melanosomes in brightfield (A, D, G, and J), the cell shape in phase contrast (B, E, H, and K), and the distribution of GFP fluorescence due primarily to unfused GFP expressed from plasmid <t>pEGFP</t> C1 (C, F, I, and L), which was coinjected to ensure accurate identification of all injected cells (see text), in dilute melanocytes microinjected with MC MV (A–C), MC MVΔD (D–F), MC MVΔF (G–I), or BR MV (J–L). Bars, 11.5 μm.
    Sali Digested Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa plasmid dna pegfp c1
    MC MV and MC MVΔD, but not MC MVΔF and BR MV, rescue melanosome distribution in dilute melanocytes. Shown is the distribution of melanosomes in brightfield (A, D, G, and J), the cell shape in phase contrast (B, E, H, and K), and the distribution of GFP fluorescence due primarily to unfused GFP expressed from plasmid <t>pEGFP</t> C1 (C, F, I, and L), which was coinjected to ensure accurate identification of all injected cells (see text), in dilute melanocytes microinjected with MC MV (A–C), MC MVΔD (D–F), MC MVΔF (G–I), or BR MV (J–L). Bars, 11.5 μm.
    Plasmid Dna Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa methuosis pegfp c1
    MC MV and MC MVΔD, but not MC MVΔF and BR MV, rescue melanosome distribution in dilute melanocytes. Shown is the distribution of melanosomes in brightfield (A, D, G, and J), the cell shape in phase contrast (B, E, H, and K), and the distribution of GFP fluorescence due primarily to unfused GFP expressed from plasmid <t>pEGFP</t> C1 (C, F, I, and L), which was coinjected to ensure accurate identification of all injected cells (see text), in dilute melanocytes microinjected with MC MV (A–C), MC MVΔD (D–F), MC MVΔF (G–I), or BR MV (J–L). Bars, 11.5 μm.
    Methuosis Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa mammalian expression vector pegfp c1
    MC MV and MC MVΔD, but not MC MVΔF and BR MV, rescue melanosome distribution in dilute melanocytes. Shown is the distribution of melanosomes in brightfield (A, D, G, and J), the cell shape in phase contrast (B, E, H, and K), and the distribution of GFP fluorescence due primarily to unfused GFP expressed from plasmid <t>pEGFP</t> C1 (C, F, I, and L), which was coinjected to ensure accurate identification of all injected cells (see text), in dilute melanocytes microinjected with MC MV (A–C), MC MVΔD (D–F), MC MVΔF (G–I), or BR MV (J–L). Bars, 11.5 μm.
    Mammalian Expression Vector Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 89/100, based on 187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa site directed mutagenesis plasmid pegfp c1
    MC MV and MC MVΔD, but not MC MVΔF and BR MV, rescue melanosome distribution in dilute melanocytes. Shown is the distribution of melanosomes in brightfield (A, D, G, and J), the cell shape in phase contrast (B, E, H, and K), and the distribution of GFP fluorescence due primarily to unfused GFP expressed from plasmid <t>pEGFP</t> C1 (C, F, I, and L), which was coinjected to ensure accurate identification of all injected cells (see text), in dilute melanocytes microinjected with MC MV (A–C), MC MVΔD (D–F), MC MVΔF (G–I), or BR MV (J–L). Bars, 11.5 μm.
    Site Directed Mutagenesis Plasmid Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa expression plasmid pegfp c1
    MC MV and MC MVΔD, but not MC MVΔF and BR MV, rescue melanosome distribution in dilute melanocytes. Shown is the distribution of melanosomes in brightfield (A, D, G, and J), the cell shape in phase contrast (B, E, H, and K), and the distribution of GFP fluorescence due primarily to unfused GFP expressed from plasmid <t>pEGFP</t> C1 (C, F, I, and L), which was coinjected to ensure accurate identification of all injected cells (see text), in dilute melanocytes microinjected with MC MV (A–C), MC MVΔD (D–F), MC MVΔF (G–I), or BR MV (J–L). Bars, 11.5 μm.
    Expression Plasmid Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa bglii hindiii digested pegfp c1
    MC MV and MC MVΔD, but not MC MVΔF and BR MV, rescue melanosome distribution in dilute melanocytes. Shown is the distribution of melanosomes in brightfield (A, D, G, and J), the cell shape in phase contrast (B, E, H, and K), and the distribution of GFP fluorescence due primarily to unfused GFP expressed from plasmid <t>pEGFP</t> C1 (C, F, I, and L), which was coinjected to ensure accurate identification of all injected cells (see text), in dilute melanocytes microinjected with MC MV (A–C), MC MVΔD (D–F), MC MVΔF (G–I), or BR MV (J–L). Bars, 11.5 μm.
    Bglii Hindiii Digested Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa plasmid encoding enhanced green fluorescent protein pegfp c1
    GFP expressions in A10 cells incubated with <t>pEGFP-C1</t> loaded NP-stents. A ) pEGFP-C1-loaded NP-stents; B ) dodecylated chitosan-coating stent (no plasmid DNA) (FITC, 100×).
    Plasmid Encoding Enhanced Green Fluorescent Protein Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa fluorescent protein pegfp c1
    Immunocytochemical detection of MKL1 and MKL2 protein in cortical neurons with synapses. ( a ) Immunostaining of mature, cortical neuronal cultures. <t>pEGFP-C1</t> vector (4 μg/well) was transfected into cortical neurons at 18 days in culture. Three days later, cells were subjected to immunostaining using anti-GFP and anti-MKL1 or anti-MKL2 antibodies. ( b ) High magnification of GFP-positive cortical neurons at 21 days in culture. Arrowheads indicate dendritic spines. ( c , d ) Synaptic staining of MKL1 and MKL2 in mature, cortical neurons. Cortical neurons at 21 days in culture were immunostained using anti-MKL1, anti-MKL2, anti-postsynaptic density (PSD)-95, and anti-synaptophysin antibodies. MKL and PSD-95 signals merged. MKL and synaptophysin signals also merged.
    Fluorescent Protein Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa mammalian expression vectors pegfp c1
    Cellular localizations of low-risk and high-risk HPV E6 in COS-1 cells. COS-1 cells were transfected with plasmids pZMZ66 (6E6E7, nt 85 to 939), pZMZ67 (11E6E7, nt 85 to 924), pZMZ69 (18E6E7, nt 103 to 967), pZMZ70 (16E6E7, nt 81 to 880), and pZMZ81 (16E6E7, nt 81 to 880 with a mutant nt 226 5′ splice site [GU to GG]) (A) and pZMZ82 (16E6, nt 81 to 559 with a mutant nt 226 5′ splice site [GU to GG]), pZMZ71 (16E6*I cDNA, nt 81 to 559), pZMZ72 (16E6*II cDNA, nt 81 to 559), pZMZ73 (16E6, nt 81 to 559), and pZMZ74 (16E7, nt 560 to 880) (B). 16E6*I and 16E6*II cDNA constructs have no coding sequences in the E6 intron region because of pre-mRNA splicing. Diagrams at the top of each panel show the insertion and position of the “E6” or “E7” coding region in mammalian expression vector <t>pEGFP-C1.</t> The individual images were collected at 6 (6E6 and 11E6) or 24 h after transfection. Plasmid pEGFP-C1 was used as a GFP control in each transfection. A fluorescence image, a differential interference contrast (DIC) image, and a merged fluorescence-DIC image for each fusion protein arepresented. (A) Localization of 6E6, 11E6, 16E6, and 18E6 translated from the inserted E6E7 coding region in COS-1 cells. 16E6 mt 5′ ss indicates the plasmid containing the 16E6E7 coding region but having a mutant 5′ splice site at nt 226 position (GU to GG), used as a transfection control for full-length 16E6, since this mutation blocks the splicing of the E6E7 pre-mRNA. The single nucleotide mutation in the 5′ splice site of 16E6E7 converts a GUA codon for valine in 16E6 to a GGA codon for glycine. (B) Localization of various 16E6 proteins translated from the inserted E6 coding region and E6*I or E6*II cDNA. 16E7 protein translated from plasmid pZMZ74 was used for comparison. + or − indicates the presence or absence of cytoplasmic (C), nuclear (N), or nucleolar (No) localization of GFP fusions. Scale bars, ∼8 μm. (C) Western blotting analysis of GFP-E6 or GFP-E7 fusions expressed from pEGFP-C1 vectors. As shown in panels A and B, all GFP fusions except for mutant E6 were included for comparison of the expected full-length E6 or E7 protein, even through the 6E6 and 11E6 coding regions do not have an intron. GFP-16E6 in lane 5, encoded from a plasmid with the 16E6E7 coding region, was included for comparison with GFP-16E6 in lane 8, encoded from a plasmid with the 16E6 coding region. Protein samples prepared from COS-1 cells transfected with the various plasmids were resolved on an SDS-12% polyacrylamide gel and blotted with anti-GFP antibody. GFP and its derived fusions are indicated above the lanes. Prestained protein markers (Bio-Rad) in kilodaltons are shown at the left.
    Mammalian Expression Vectors Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    TaKaRa cloning vectors pegfp c1
    Cellular localizations of low-risk and high-risk HPV E6 in COS-1 cells. COS-1 cells were transfected with plasmids pZMZ66 (6E6E7, nt 85 to 939), pZMZ67 (11E6E7, nt 85 to 924), pZMZ69 (18E6E7, nt 103 to 967), pZMZ70 (16E6E7, nt 81 to 880), and pZMZ81 (16E6E7, nt 81 to 880 with a mutant nt 226 5′ splice site [GU to GG]) (A) and pZMZ82 (16E6, nt 81 to 559 with a mutant nt 226 5′ splice site [GU to GG]), pZMZ71 (16E6*I cDNA, nt 81 to 559), pZMZ72 (16E6*II cDNA, nt 81 to 559), pZMZ73 (16E6, nt 81 to 559), and pZMZ74 (16E7, nt 560 to 880) (B). 16E6*I and 16E6*II cDNA constructs have no coding sequences in the E6 intron region because of pre-mRNA splicing. Diagrams at the top of each panel show the insertion and position of the “E6” or “E7” coding region in mammalian expression vector <t>pEGFP-C1.</t> The individual images were collected at 6 (6E6 and 11E6) or 24 h after transfection. Plasmid pEGFP-C1 was used as a GFP control in each transfection. A fluorescence image, a differential interference contrast (DIC) image, and a merged fluorescence-DIC image for each fusion protein arepresented. (A) Localization of 6E6, 11E6, 16E6, and 18E6 translated from the inserted E6E7 coding region in COS-1 cells. 16E6 mt 5′ ss indicates the plasmid containing the 16E6E7 coding region but having a mutant 5′ splice site at nt 226 position (GU to GG), used as a transfection control for full-length 16E6, since this mutation blocks the splicing of the E6E7 pre-mRNA. The single nucleotide mutation in the 5′ splice site of 16E6E7 converts a GUA codon for valine in 16E6 to a GGA codon for glycine. (B) Localization of various 16E6 proteins translated from the inserted E6 coding region and E6*I or E6*II cDNA. 16E7 protein translated from plasmid pZMZ74 was used for comparison. + or − indicates the presence or absence of cytoplasmic (C), nuclear (N), or nucleolar (No) localization of GFP fusions. Scale bars, ∼8 μm. (C) Western blotting analysis of GFP-E6 or GFP-E7 fusions expressed from pEGFP-C1 vectors. As shown in panels A and B, all GFP fusions except for mutant E6 were included for comparison of the expected full-length E6 or E7 protein, even through the 6E6 and 11E6 coding regions do not have an intron. GFP-16E6 in lane 5, encoded from a plasmid with the 16E6E7 coding region, was included for comparison with GFP-16E6 in lane 8, encoded from a plasmid with the 16E6 coding region. Protein samples prepared from COS-1 cells transfected with the various plasmids were resolved on an SDS-12% polyacrylamide gel and blotted with anti-GFP antibody. GFP and its derived fusions are indicated above the lanes. Prestained protein markers (Bio-Rad) in kilodaltons are shown at the left.
    Cloning Vectors Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 80/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa kpni bamhi digested pegfp c1
    Cellular localizations of low-risk and high-risk HPV E6 in COS-1 cells. COS-1 cells were transfected with plasmids pZMZ66 (6E6E7, nt 85 to 939), pZMZ67 (11E6E7, nt 85 to 924), pZMZ69 (18E6E7, nt 103 to 967), pZMZ70 (16E6E7, nt 81 to 880), and pZMZ81 (16E6E7, nt 81 to 880 with a mutant nt 226 5′ splice site [GU to GG]) (A) and pZMZ82 (16E6, nt 81 to 559 with a mutant nt 226 5′ splice site [GU to GG]), pZMZ71 (16E6*I cDNA, nt 81 to 559), pZMZ72 (16E6*II cDNA, nt 81 to 559), pZMZ73 (16E6, nt 81 to 559), and pZMZ74 (16E7, nt 560 to 880) (B). 16E6*I and 16E6*II cDNA constructs have no coding sequences in the E6 intron region because of pre-mRNA splicing. Diagrams at the top of each panel show the insertion and position of the “E6” or “E7” coding region in mammalian expression vector <t>pEGFP-C1.</t> The individual images were collected at 6 (6E6 and 11E6) or 24 h after transfection. Plasmid pEGFP-C1 was used as a GFP control in each transfection. A fluorescence image, a differential interference contrast (DIC) image, and a merged fluorescence-DIC image for each fusion protein arepresented. (A) Localization of 6E6, 11E6, 16E6, and 18E6 translated from the inserted E6E7 coding region in COS-1 cells. 16E6 mt 5′ ss indicates the plasmid containing the 16E6E7 coding region but having a mutant 5′ splice site at nt 226 position (GU to GG), used as a transfection control for full-length 16E6, since this mutation blocks the splicing of the E6E7 pre-mRNA. The single nucleotide mutation in the 5′ splice site of 16E6E7 converts a GUA codon for valine in 16E6 to a GGA codon for glycine. (B) Localization of various 16E6 proteins translated from the inserted E6 coding region and E6*I or E6*II cDNA. 16E7 protein translated from plasmid pZMZ74 was used for comparison. + or − indicates the presence or absence of cytoplasmic (C), nuclear (N), or nucleolar (No) localization of GFP fusions. Scale bars, ∼8 μm. (C) Western blotting analysis of GFP-E6 or GFP-E7 fusions expressed from pEGFP-C1 vectors. As shown in panels A and B, all GFP fusions except for mutant E6 were included for comparison of the expected full-length E6 or E7 protein, even through the 6E6 and 11E6 coding regions do not have an intron. GFP-16E6 in lane 5, encoded from a plasmid with the 16E6E7 coding region, was included for comparison with GFP-16E6 in lane 8, encoded from a plasmid with the 16E6 coding region. Protein samples prepared from COS-1 cells transfected with the various plasmids were resolved on an SDS-12% polyacrylamide gel and blotted with anti-GFP antibody. GFP and its derived fusions are indicated above the lanes. Prestained protein markers (Bio-Rad) in kilodaltons are shown at the left.
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    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: To get further insights into possible causes of inhibitory effects of pEGFP-C1, we re-examined deep sequencing data searching for any transcriptome features unique to 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: To get further insights into possible causes of inhibitory effects of pEGFP-C1, we re-examined deep sequencing data searching for any transcriptome features unique to pEGFP-C1.

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

    Rab5 and Rabankyrin5 overexpression increases macropinosome formation. The distribution of Rab5, Rabankyrin-5 (A) or Rab5(Q79L) (B) in green relative to dextran-positive macropinosomes (red) Scale bar = 10 µm. (C) HEK-Flp-In cells were transiently transfected with pEGFP-C1, pEGFP-Rab5, pEGFP-Rab5-Q79L and pEYFP-Rabankyrin-5. The mean number of macropinosomes/100 transfected cells was quantitated over 3 replicates of 500 transfected cells for each condition. * denotes statistical significance (p

    Journal: PLoS ONE

    Article Title: The SNX-PX-BAR Family in Macropinocytosis: The Regulation of Macropinosome Formation by SNX-PX-BAR Proteins

    doi: 10.1371/journal.pone.0013763

    Figure Lengend Snippet: Rab5 and Rabankyrin5 overexpression increases macropinosome formation. The distribution of Rab5, Rabankyrin-5 (A) or Rab5(Q79L) (B) in green relative to dextran-positive macropinosomes (red) Scale bar = 10 µm. (C) HEK-Flp-In cells were transiently transfected with pEGFP-C1, pEGFP-Rab5, pEGFP-Rab5-Q79L and pEYFP-Rabankyrin-5. The mean number of macropinosomes/100 transfected cells was quantitated over 3 replicates of 500 transfected cells for each condition. * denotes statistical significance (p

    Article Snippet: The construct pEGFP-C1 was obtained from Clontech. pEGFP-Rab5 , pEGFP-Rab5Q79L , and pEYFP-Rabankyrin 5 , are as described previously. pIRES2-EGFP-PTEN and pIRES2-EGFP-PTEN(G129E) were generated by subcloning the open-reading frames of PTEN and PTEN(G129E) into pIRES2-EGFP using BamHI and EcoRI. pEGFP-SNX1, pEGFP-SNX2, pEGFP-SNX4, pEGFP-SNX5, pEGFP-SNX6, pEGFP-SNX7, pEGFP-SNX8, pEGFP-SNX9, pEGFP-SNX18, pEGFP-SNX30, pEGFP-SNX32, and pEGFP-SNX33 were generated by amplifying their full length open reading frame by polymerase chain reaction (PCR) and the resulting PCR products cloned into pEGFP-C1. pmCherry-SNX18 was generated by amplifying the full length open reading frame of SNX18 and cloning the resulting PCR product into pmCherry-C1. pEGFP-ΔSH3-SNX18 was generated by amplifying residues 61-615 of SNX18 and cloning the resulting PCR product into pEGFP-C1.

    Techniques: Over Expression, Transfection

    Macropinosome formation screening assay validation. A: 24 hours post transfection, HEK-Flp-In cell monolayers were pulsed for 5 minutes with 100 µg/mL dextran (10,000 MW) conjugated to tetramethylrhodamine (dextran-TR) at 37°C. The samples were then washed in 4°C PBS, fixed in 4% PFA and imaged and processed as described in Materials and Methods . Briefly Z-stack images comprising of 3×5 µm Z slices were merged into a single RGB image of transfected cells (green) stained with dextran-TR (red) (Ai). The red channel from the RGB image was isolated and converted to an 8-bit grayscale image (Aii). Dextran-positive macropinosomes were selected based on size ( > 0.5 µm in diameter) and fluorescent intensity ( > 100). Selected macropinosomes are shown in the foreground as black (Aiii). This binary image was then converted to a mask and superimposed onto the Green channel of the original RGB image to measure the green fluorescent intensity of the area occupied by each macropinosome in the image (Aiv). The particles with green fluorescence intensity higher than background signal ( > 20) were considered to be macropinosomes within a transfected cell, represented in green. Red particles represent discarded macropinosomes determined to be outside of a transfected cell. Scale = 10 µm. B, C, D: HEK-Flp-In cell monolayers were either serum-starved for 16 hours and treated with dextran-TR in the presence or absence of 100 ng/mL EGF for 5 minutes at 37°C (B), treated with 1 mM amiloride or carrier (0.6% Methanol) for 30 minutes at 37°C before pulsing with dextran-TR (C), or transiently transfected with pEGFP-C1 or pEGFP-SNX5 before pulsing with dextran-TR (D). The samples in each case were assayed for macropinosome formation as described in Materials and Methods , quantitating the mean number of macropinosomes/100 transfected cells over 3 replicates of 500 transfected cells for each condition. * denotes statistical significance (p

    Journal: PLoS ONE

    Article Title: The SNX-PX-BAR Family in Macropinocytosis: The Regulation of Macropinosome Formation by SNX-PX-BAR Proteins

    doi: 10.1371/journal.pone.0013763

    Figure Lengend Snippet: Macropinosome formation screening assay validation. A: 24 hours post transfection, HEK-Flp-In cell monolayers were pulsed for 5 minutes with 100 µg/mL dextran (10,000 MW) conjugated to tetramethylrhodamine (dextran-TR) at 37°C. The samples were then washed in 4°C PBS, fixed in 4% PFA and imaged and processed as described in Materials and Methods . Briefly Z-stack images comprising of 3×5 µm Z slices were merged into a single RGB image of transfected cells (green) stained with dextran-TR (red) (Ai). The red channel from the RGB image was isolated and converted to an 8-bit grayscale image (Aii). Dextran-positive macropinosomes were selected based on size ( > 0.5 µm in diameter) and fluorescent intensity ( > 100). Selected macropinosomes are shown in the foreground as black (Aiii). This binary image was then converted to a mask and superimposed onto the Green channel of the original RGB image to measure the green fluorescent intensity of the area occupied by each macropinosome in the image (Aiv). The particles with green fluorescence intensity higher than background signal ( > 20) were considered to be macropinosomes within a transfected cell, represented in green. Red particles represent discarded macropinosomes determined to be outside of a transfected cell. Scale = 10 µm. B, C, D: HEK-Flp-In cell monolayers were either serum-starved for 16 hours and treated with dextran-TR in the presence or absence of 100 ng/mL EGF for 5 minutes at 37°C (B), treated with 1 mM amiloride or carrier (0.6% Methanol) for 30 minutes at 37°C before pulsing with dextran-TR (C), or transiently transfected with pEGFP-C1 or pEGFP-SNX5 before pulsing with dextran-TR (D). The samples in each case were assayed for macropinosome formation as described in Materials and Methods , quantitating the mean number of macropinosomes/100 transfected cells over 3 replicates of 500 transfected cells for each condition. * denotes statistical significance (p

    Article Snippet: The construct pEGFP-C1 was obtained from Clontech. pEGFP-Rab5 , pEGFP-Rab5Q79L , and pEYFP-Rabankyrin 5 , are as described previously. pIRES2-EGFP-PTEN and pIRES2-EGFP-PTEN(G129E) were generated by subcloning the open-reading frames of PTEN and PTEN(G129E) into pIRES2-EGFP using BamHI and EcoRI. pEGFP-SNX1, pEGFP-SNX2, pEGFP-SNX4, pEGFP-SNX5, pEGFP-SNX6, pEGFP-SNX7, pEGFP-SNX8, pEGFP-SNX9, pEGFP-SNX18, pEGFP-SNX30, pEGFP-SNX32, and pEGFP-SNX33 were generated by amplifying their full length open reading frame by polymerase chain reaction (PCR) and the resulting PCR products cloned into pEGFP-C1. pmCherry-SNX18 was generated by amplifying the full length open reading frame of SNX18 and cloning the resulting PCR product into pmCherry-C1. pEGFP-ΔSH3-SNX18 was generated by amplifying residues 61-615 of SNX18 and cloning the resulting PCR product into pEGFP-C1.

    Techniques: Screening Assay, Transfection, Staining, Isolation, Fluorescence

    Confocal microscopic analysis of M2 expression. A20 cells were transfected with either pEGFP-C1 (A to C) or pEGFP-C1/M2 (D to I) and after 48 h were either fixed, permeabilized, and then counterstained with PI or stained with TRITC-conjugated antibody to MHC class II antigens and then fixed. Cells were then visualized using a Leica TCNTS confocal microscope with combinations of laser light wavelength and band filter specific for EGFP (488 and 525/50 nm [A, D, and G]) or for PI and TRITC (568 and 600/30 nm [B, E, and H]). Image analysis with Leica software enabled the overlaying of these two images (C, F, and I). All images are shown at the same magnification.

    Journal: Journal of Virology

    Article Title: Murid Herpesvirus 4 Strain 68 M2 Protein Is a B-Cell-Associated Antigen Important for Latency but Not Lymphocytosis

    doi: 10.1128/JVI.77.17.9700-9709.2003

    Figure Lengend Snippet: Confocal microscopic analysis of M2 expression. A20 cells were transfected with either pEGFP-C1 (A to C) or pEGFP-C1/M2 (D to I) and after 48 h were either fixed, permeabilized, and then counterstained with PI or stained with TRITC-conjugated antibody to MHC class II antigens and then fixed. Cells were then visualized using a Leica TCNTS confocal microscope with combinations of laser light wavelength and band filter specific for EGFP (488 and 525/50 nm [A, D, and G]) or for PI and TRITC (568 and 600/30 nm [B, E, and H]). Image analysis with Leica software enabled the overlaying of these two images (C, F, and I). All images are shown at the same magnification.

    Article Snippet: Because of the lack of an adequate M2-specific antibody, M2 was expressed as either an N- or C-terminal fusion with enhanced green fluorescent protein (EGFP) by using the vectors pEGFP-C1 and pEGFP-N1 (Clontech) to aid in its detection.

    Techniques: Expressing, Transfection, Staining, Microscopy, Software

    MC MV and MC MVΔD, but not MC MVΔF and BR MV, rescue melanosome distribution in dilute melanocytes. Shown is the distribution of melanosomes in brightfield (A, D, G, and J), the cell shape in phase contrast (B, E, H, and K), and the distribution of GFP fluorescence due primarily to unfused GFP expressed from plasmid pEGFP C1 (C, F, I, and L), which was coinjected to ensure accurate identification of all injected cells (see text), in dilute melanocytes microinjected with MC MV (A–C), MC MVΔD (D–F), MC MVΔF (G–I), or BR MV (J–L). Bars, 11.5 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Rab27a Is an Essential Component of Melanosome Receptor for Myosin Va

    doi: 10.1091/mbc.01-12-0595

    Figure Lengend Snippet: MC MV and MC MVΔD, but not MC MVΔF and BR MV, rescue melanosome distribution in dilute melanocytes. Shown is the distribution of melanosomes in brightfield (A, D, G, and J), the cell shape in phase contrast (B, E, H, and K), and the distribution of GFP fluorescence due primarily to unfused GFP expressed from plasmid pEGFP C1 (C, F, I, and L), which was coinjected to ensure accurate identification of all injected cells (see text), in dilute melanocytes microinjected with MC MV (A–C), MC MVΔD (D–F), MC MVΔF (G–I), or BR MV (J–L). Bars, 11.5 μm.

    Article Snippet: The resulting fragment was digested with Bam HI and Eco RI and cloned into plasmid pEGFP C1 that had been cut with Bgl II and Eco RI.

    Techniques: Fluorescence, Plasmid Preparation, Injection

    Overexpressed GJA8 protein modulated the level of LC3 and P62 in HLE cells. a HLE cells stained for GJA8 (red) and LC3 (green) in the NC and Sta2h groups. b Western blot with LC3-I/II in NC and Sta2h groups in HLE cells. c The average level of LC3-II/Actin of three independent experiments. d Transfected with peGFP-C1-GJA8 in the NC and Sta2h groups, HLE cells stained for LC3 (red). e Mean number of LC3 puncta for each treatment ( n = 3 wells, 3 independent experiments, > 50 cells per experiment). f HLE cells for corresponding treatment and transfected with peGFP-C1-GJA8 for 0ug or 1ug or 2ug, testing the expression of P62 and α-Tubulin. g The average level of P62/α-Tubulin of three independent experiments in Sta4h/Sta2h/NC group. h The average level of P62/α-Tubulin of three independent experiments transfected with peGFP-C1-GJA8 for 0ug or 1ug or 2ug in NC group. All values are represented as the mean + SEM; * p

    Journal: Human Genetics

    Article Title: The impact of GJA8 SNPs on susceptibility to age-related cataract

    doi: 10.1007/s00439-018-1945-5

    Figure Lengend Snippet: Overexpressed GJA8 protein modulated the level of LC3 and P62 in HLE cells. a HLE cells stained for GJA8 (red) and LC3 (green) in the NC and Sta2h groups. b Western blot with LC3-I/II in NC and Sta2h groups in HLE cells. c The average level of LC3-II/Actin of three independent experiments. d Transfected with peGFP-C1-GJA8 in the NC and Sta2h groups, HLE cells stained for LC3 (red). e Mean number of LC3 puncta for each treatment ( n = 3 wells, 3 independent experiments, > 50 cells per experiment). f HLE cells for corresponding treatment and transfected with peGFP-C1-GJA8 for 0ug or 1ug or 2ug, testing the expression of P62 and α-Tubulin. g The average level of P62/α-Tubulin of three independent experiments in Sta4h/Sta2h/NC group. h The average level of P62/α-Tubulin of three independent experiments transfected with peGFP-C1-GJA8 for 0ug or 1ug or 2ug in NC group. All values are represented as the mean + SEM; * p

    Article Snippet: The PCR products and vector pEGFP-C1 were digested by Xho I and Eco rI (Takara, Japan).

    Techniques: Staining, Western Blot, Transfection, Expressing

    GFP expressions in A10 cells incubated with pEGFP-C1 loaded NP-stents. A ) pEGFP-C1-loaded NP-stents; B ) dodecylated chitosan-coating stent (no plasmid DNA) (FITC, 100×).

    Journal: International Journal of Nanomedicine

    Article Title: Local gene delivery via endovascular stents coated with dodecylated chitosan-plasmid DNA nanoparticles

    doi: 10.2147/IJN.S14358

    Figure Lengend Snippet: GFP expressions in A10 cells incubated with pEGFP-C1 loaded NP-stents. A ) pEGFP-C1-loaded NP-stents; B ) dodecylated chitosan-coating stent (no plasmid DNA) (FITC, 100×).

    Article Snippet: Plasmid encoding enhanced green fluorescent protein (pEGFP-C1) was purchased from Clontech (Palo Alto, CA).

    Techniques: Incubation, Plasmid Preparation

    Rabbit CCA results demonstrating cells expressing GFP mediated by pEGFP-C1-loaded NP stents in vivo.

    Journal: International Journal of Nanomedicine

    Article Title: Local gene delivery via endovascular stents coated with dodecylated chitosan-plasmid DNA nanoparticles

    doi: 10.2147/IJN.S14358

    Figure Lengend Snippet: Rabbit CCA results demonstrating cells expressing GFP mediated by pEGFP-C1-loaded NP stents in vivo.

    Article Snippet: Plasmid encoding enhanced green fluorescent protein (pEGFP-C1) was purchased from Clontech (Palo Alto, CA).

    Techniques: Expressing, In Vivo

    Immunocytochemical detection of MKL1 and MKL2 protein in cortical neurons with synapses. ( a ) Immunostaining of mature, cortical neuronal cultures. pEGFP-C1 vector (4 μg/well) was transfected into cortical neurons at 18 days in culture. Three days later, cells were subjected to immunostaining using anti-GFP and anti-MKL1 or anti-MKL2 antibodies. ( b ) High magnification of GFP-positive cortical neurons at 21 days in culture. Arrowheads indicate dendritic spines. ( c , d ) Synaptic staining of MKL1 and MKL2 in mature, cortical neurons. Cortical neurons at 21 days in culture were immunostained using anti-MKL1, anti-MKL2, anti-postsynaptic density (PSD)-95, and anti-synaptophysin antibodies. MKL and PSD-95 signals merged. MKL and synaptophysin signals also merged.

    Journal: Scientific Reports

    Article Title: Synaptic localisation of SRF coactivators, MKL1 and MKL2, and their role in dendritic spine morphology

    doi: 10.1038/s41598-017-18905-7

    Figure Lengend Snippet: Immunocytochemical detection of MKL1 and MKL2 protein in cortical neurons with synapses. ( a ) Immunostaining of mature, cortical neuronal cultures. pEGFP-C1 vector (4 μg/well) was transfected into cortical neurons at 18 days in culture. Three days later, cells were subjected to immunostaining using anti-GFP and anti-MKL1 or anti-MKL2 antibodies. ( b ) High magnification of GFP-positive cortical neurons at 21 days in culture. Arrowheads indicate dendritic spines. ( c , d ) Synaptic staining of MKL1 and MKL2 in mature, cortical neurons. Cortical neurons at 21 days in culture were immunostained using anti-MKL1, anti-MKL2, anti-postsynaptic density (PSD)-95, and anti-synaptophysin antibodies. MKL and PSD-95 signals merged. MKL and synaptophysin signals also merged.

    Article Snippet: The expression vector for enhanced green fluorescent protein (pEGFP-C1) was purchased from Clontech.

    Techniques: Immunostaining, Plasmid Preparation, Transfection, Staining

    Effect of MKL1 or MKL2 knock-down on dendritic spine morphology and density in cortical neurons. ( a ) Spine morphology of cortical neurons transfected with pEGFP-C1 (1 μg/well) and shR-luc, sh MKL1 , or sh MKL2 (1 μg/well). Transfection was performed at 16 days in culture. Neurons (21 days in culture) were immunostained using anti-GFP antibody. ( b , c ) Spine morphology (m and s, mushroom or stubby; t and f, thin or filopodia; irregular) and spine density. Graphs show mean ± S.D. from seven independent experiments. The statistical significance of differences (vs. shR-luc) was analysed by ANOVA with the Tukey–Kramer test. * p

    Journal: Scientific Reports

    Article Title: Synaptic localisation of SRF coactivators, MKL1 and MKL2, and their role in dendritic spine morphology

    doi: 10.1038/s41598-017-18905-7

    Figure Lengend Snippet: Effect of MKL1 or MKL2 knock-down on dendritic spine morphology and density in cortical neurons. ( a ) Spine morphology of cortical neurons transfected with pEGFP-C1 (1 μg/well) and shR-luc, sh MKL1 , or sh MKL2 (1 μg/well). Transfection was performed at 16 days in culture. Neurons (21 days in culture) were immunostained using anti-GFP antibody. ( b , c ) Spine morphology (m and s, mushroom or stubby; t and f, thin or filopodia; irregular) and spine density. Graphs show mean ± S.D. from seven independent experiments. The statistical significance of differences (vs. shR-luc) was analysed by ANOVA with the Tukey–Kramer test. * p

    Article Snippet: The expression vector for enhanced green fluorescent protein (pEGFP-C1) was purchased from Clontech.

    Techniques: Transfection

    Specific detection of MKL1 and MKL2 by anti-MKL1 and anti-MKL2 antibodies. ( a ) pEGFP-C1 vector and pFLAG-CMV2 vector (FLAG-empty) or pFLAG-CMV2-MKL1 vector (FLAG-MKL1), pCMV-HA vector (HA-empty) or pCMV-HA-MKL2 vector (HA-MKL2) were co-transfected into NIH3T3 cells. Cell lysates were lysed 24 hours after transfection. Anti-MKL1 (MKL1) and anti-MKL2 (MKL2) antibody were used for detection of exogenous and endogenous MKL1 and MKL2, respectively. Anti-FLAG and anti-HA antibodies were used for detection of exogenous MKL1 and MKL2, respectively. Anti-α-tubulin and anti-GFP antibodies were used as internal (loading) controls. ( b ) Detection of endogenous MKL1 and MKL2 in cortical neurons. Left-most lanes: cell lysates from NIH3T3 cells overexpressing FLAG-MKL1 and HA-MKL2. Lane 2: cell lysates from untransfected NIH3T3 cells. Lane 3: cell lysates from rat cortical neurons at 7 days in culture. Full-length blots are presented in Supplementary Figure S5 .

    Journal: Scientific Reports

    Article Title: Synaptic localisation of SRF coactivators, MKL1 and MKL2, and their role in dendritic spine morphology

    doi: 10.1038/s41598-017-18905-7

    Figure Lengend Snippet: Specific detection of MKL1 and MKL2 by anti-MKL1 and anti-MKL2 antibodies. ( a ) pEGFP-C1 vector and pFLAG-CMV2 vector (FLAG-empty) or pFLAG-CMV2-MKL1 vector (FLAG-MKL1), pCMV-HA vector (HA-empty) or pCMV-HA-MKL2 vector (HA-MKL2) were co-transfected into NIH3T3 cells. Cell lysates were lysed 24 hours after transfection. Anti-MKL1 (MKL1) and anti-MKL2 (MKL2) antibody were used for detection of exogenous and endogenous MKL1 and MKL2, respectively. Anti-FLAG and anti-HA antibodies were used for detection of exogenous MKL1 and MKL2, respectively. Anti-α-tubulin and anti-GFP antibodies were used as internal (loading) controls. ( b ) Detection of endogenous MKL1 and MKL2 in cortical neurons. Left-most lanes: cell lysates from NIH3T3 cells overexpressing FLAG-MKL1 and HA-MKL2. Lane 2: cell lysates from untransfected NIH3T3 cells. Lane 3: cell lysates from rat cortical neurons at 7 days in culture. Full-length blots are presented in Supplementary Figure S5 .

    Article Snippet: The expression vector for enhanced green fluorescent protein (pEGFP-C1) was purchased from Clontech.

    Techniques: Plasmid Preparation, Transfection

    Cellular localizations of low-risk and high-risk HPV E6 in COS-1 cells. COS-1 cells were transfected with plasmids pZMZ66 (6E6E7, nt 85 to 939), pZMZ67 (11E6E7, nt 85 to 924), pZMZ69 (18E6E7, nt 103 to 967), pZMZ70 (16E6E7, nt 81 to 880), and pZMZ81 (16E6E7, nt 81 to 880 with a mutant nt 226 5′ splice site [GU to GG]) (A) and pZMZ82 (16E6, nt 81 to 559 with a mutant nt 226 5′ splice site [GU to GG]), pZMZ71 (16E6*I cDNA, nt 81 to 559), pZMZ72 (16E6*II cDNA, nt 81 to 559), pZMZ73 (16E6, nt 81 to 559), and pZMZ74 (16E7, nt 560 to 880) (B). 16E6*I and 16E6*II cDNA constructs have no coding sequences in the E6 intron region because of pre-mRNA splicing. Diagrams at the top of each panel show the insertion and position of the “E6” or “E7” coding region in mammalian expression vector pEGFP-C1. The individual images were collected at 6 (6E6 and 11E6) or 24 h after transfection. Plasmid pEGFP-C1 was used as a GFP control in each transfection. A fluorescence image, a differential interference contrast (DIC) image, and a merged fluorescence-DIC image for each fusion protein arepresented. (A) Localization of 6E6, 11E6, 16E6, and 18E6 translated from the inserted E6E7 coding region in COS-1 cells. 16E6 mt 5′ ss indicates the plasmid containing the 16E6E7 coding region but having a mutant 5′ splice site at nt 226 position (GU to GG), used as a transfection control for full-length 16E6, since this mutation blocks the splicing of the E6E7 pre-mRNA. The single nucleotide mutation in the 5′ splice site of 16E6E7 converts a GUA codon for valine in 16E6 to a GGA codon for glycine. (B) Localization of various 16E6 proteins translated from the inserted E6 coding region and E6*I or E6*II cDNA. 16E7 protein translated from plasmid pZMZ74 was used for comparison. + or − indicates the presence or absence of cytoplasmic (C), nuclear (N), or nucleolar (No) localization of GFP fusions. Scale bars, ∼8 μm. (C) Western blotting analysis of GFP-E6 or GFP-E7 fusions expressed from pEGFP-C1 vectors. As shown in panels A and B, all GFP fusions except for mutant E6 were included for comparison of the expected full-length E6 or E7 protein, even through the 6E6 and 11E6 coding regions do not have an intron. GFP-16E6 in lane 5, encoded from a plasmid with the 16E6E7 coding region, was included for comparison with GFP-16E6 in lane 8, encoded from a plasmid with the 16E6 coding region. Protein samples prepared from COS-1 cells transfected with the various plasmids were resolved on an SDS-12% polyacrylamide gel and blotted with anti-GFP antibody. GFP and its derived fusions are indicated above the lanes. Prestained protein markers (Bio-Rad) in kilodaltons are shown at the left.

    Journal: Journal of Virology

    Article Title: Signals That Dictate Nuclear Localization of Human Papillomavirus Type 16 Oncoprotein E6 in Living Cells

    doi: 10.1128/JVI.77.24.13232-13247.2003

    Figure Lengend Snippet: Cellular localizations of low-risk and high-risk HPV E6 in COS-1 cells. COS-1 cells were transfected with plasmids pZMZ66 (6E6E7, nt 85 to 939), pZMZ67 (11E6E7, nt 85 to 924), pZMZ69 (18E6E7, nt 103 to 967), pZMZ70 (16E6E7, nt 81 to 880), and pZMZ81 (16E6E7, nt 81 to 880 with a mutant nt 226 5′ splice site [GU to GG]) (A) and pZMZ82 (16E6, nt 81 to 559 with a mutant nt 226 5′ splice site [GU to GG]), pZMZ71 (16E6*I cDNA, nt 81 to 559), pZMZ72 (16E6*II cDNA, nt 81 to 559), pZMZ73 (16E6, nt 81 to 559), and pZMZ74 (16E7, nt 560 to 880) (B). 16E6*I and 16E6*II cDNA constructs have no coding sequences in the E6 intron region because of pre-mRNA splicing. Diagrams at the top of each panel show the insertion and position of the “E6” or “E7” coding region in mammalian expression vector pEGFP-C1. The individual images were collected at 6 (6E6 and 11E6) or 24 h after transfection. Plasmid pEGFP-C1 was used as a GFP control in each transfection. A fluorescence image, a differential interference contrast (DIC) image, and a merged fluorescence-DIC image for each fusion protein arepresented. (A) Localization of 6E6, 11E6, 16E6, and 18E6 translated from the inserted E6E7 coding region in COS-1 cells. 16E6 mt 5′ ss indicates the plasmid containing the 16E6E7 coding region but having a mutant 5′ splice site at nt 226 position (GU to GG), used as a transfection control for full-length 16E6, since this mutation blocks the splicing of the E6E7 pre-mRNA. The single nucleotide mutation in the 5′ splice site of 16E6E7 converts a GUA codon for valine in 16E6 to a GGA codon for glycine. (B) Localization of various 16E6 proteins translated from the inserted E6 coding region and E6*I or E6*II cDNA. 16E7 protein translated from plasmid pZMZ74 was used for comparison. + or − indicates the presence or absence of cytoplasmic (C), nuclear (N), or nucleolar (No) localization of GFP fusions. Scale bars, ∼8 μm. (C) Western blotting analysis of GFP-E6 or GFP-E7 fusions expressed from pEGFP-C1 vectors. As shown in panels A and B, all GFP fusions except for mutant E6 were included for comparison of the expected full-length E6 or E7 protein, even through the 6E6 and 11E6 coding regions do not have an intron. GFP-16E6 in lane 5, encoded from a plasmid with the 16E6E7 coding region, was included for comparison with GFP-16E6 in lane 8, encoded from a plasmid with the 16E6 coding region. Protein samples prepared from COS-1 cells transfected with the various plasmids were resolved on an SDS-12% polyacrylamide gel and blotted with anti-GFP antibody. GFP and its derived fusions are indicated above the lanes. Prestained protein markers (Bio-Rad) in kilodaltons are shown at the left.

    Article Snippet: Mammalian expression vectors pEGFP-C1 and pEGFP-N1 were purchased from Clontech (Palo Alto, Calif.).

    Techniques: Transfection, Mutagenesis, Construct, Expressing, Plasmid Preparation, Fluorescence, Western Blot, Derivative Assay