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  • 93
    TaKaRa hela tet on cells
    CENP-T promotes KMN kinetochore targeting through CENP-H-I-K. (A) <t>HeLa</t> <t>Tet-On</t> cells were mock transfected or transfected with siCENP-H, treated with thymidine for 14 h, released into nocodazole-containing medium for 12 h, and treated with or without ZM for 2 h. Their mitotic index was determined by flow cytometry. Means and SDs (error bars) are shown ( n = 3 independent experiments). (B) HeLa cells were transfected with the indicated plasmids and siRNAs (siCC, siCENP-C; siCC+CH, siCENP-C+siCENP-H), and treated with nocodazole. Their mitotic index (mean ± SD [error bars], n = 3) was quantified by flow cytometry. (C and D) Nocodazole-treated mitotic HeLa cells transfected with siCENP-H were further incubated with MG132 (NM) or with both MG132 and ZM (NM+Z), and stained with the indicated antibodies and DAPI. Boxed regions of merged images were magnified and shown in the rightmost column. The relative kinetochore intensities (mean ± SD, n = 400) in certain channels were quantified and shown. Bars, 5 µm (1 µm for magnified images). (E) Recombinant Ndc80C was preincubated with or without recombinant Mis12C, immobilized on beads, and incubated with 35 S-labeled CENP-H-I-K. Bound proteins and input were separated by SDS-PAGE, stained with CBB (left), and analyzed with a phosphorimager (right). CENP-H and -K co-migrate. The asterisk indicates a CENP-K fragment. Broken lines indicate that intervening lanes have been spliced out.
    Hela Tet On Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    TaKaRa tet on 3g inducible expression system
    Endogenous mitofusins locate on the mitochondrion-peroxisome contacting sites. a Immunofluorescence images of endogenous MFN2. Confocal microscopy analysis of HeLa cells <t>expressing</t> COX4-EGFP (mitochondria), immunostained for MFN2 (Alexa Fluor 555) and peroxisomal protein ABCD3 (Alexa Fluor 647). Scale bars: 5 μm. b The zoomed-in images of the white box in ( a ). White arrows point the places where ABCD3 overlaps with MFN. Scale bars: 1 μm. c Histograms display measured fluorescence intensity along the white line in the merge panels in b , with the cyan line represents mitochondria, the magenta line represents endogenous MFN2, and the red line represents peroxisome. d – f Immunofluorescence images and analysis of endogenous MFN1. Similar experiments were performed as in ( a – c ).
    Tet On 3g Inducible Expression System, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tet on 3g inducible expression system/product/TaKaRa
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tet on 3g inducible expression system - by Bioz Stars, 2022-08
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    94
    TaKaRa retro x tet on 3g inducible expression system
    Endogenous mitofusins locate on the mitochondrion-peroxisome contacting sites. a Immunofluorescence images of endogenous MFN2. Confocal microscopy analysis of HeLa cells <t>expressing</t> COX4-EGFP (mitochondria), immunostained for MFN2 (Alexa Fluor 555) and peroxisomal protein ABCD3 (Alexa Fluor 647). Scale bars: 5 μm. b The zoomed-in images of the white box in ( a ). White arrows point the places where ABCD3 overlaps with MFN. Scale bars: 1 μm. c Histograms display measured fluorescence intensity along the white line in the merge panels in b , with the cyan line represents mitochondria, the magenta line represents endogenous MFN2, and the red line represents peroxisome. d – f Immunofluorescence images and analysis of endogenous MFN1. Similar experiments were performed as in ( a – c ).
    Retro X Tet On 3g Inducible Expression System, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    CENP-T promotes KMN kinetochore targeting through CENP-H-I-K. (A) HeLa Tet-On cells were mock transfected or transfected with siCENP-H, treated with thymidine for 14 h, released into nocodazole-containing medium for 12 h, and treated with or without ZM for 2 h. Their mitotic index was determined by flow cytometry. Means and SDs (error bars) are shown ( n = 3 independent experiments). (B) HeLa cells were transfected with the indicated plasmids and siRNAs (siCC, siCENP-C; siCC+CH, siCENP-C+siCENP-H), and treated with nocodazole. Their mitotic index (mean ± SD [error bars], n = 3) was quantified by flow cytometry. (C and D) Nocodazole-treated mitotic HeLa cells transfected with siCENP-H were further incubated with MG132 (NM) or with both MG132 and ZM (NM+Z), and stained with the indicated antibodies and DAPI. Boxed regions of merged images were magnified and shown in the rightmost column. The relative kinetochore intensities (mean ± SD, n = 400) in certain channels were quantified and shown. Bars, 5 µm (1 µm for magnified images). (E) Recombinant Ndc80C was preincubated with or without recombinant Mis12C, immobilized on beads, and incubated with 35 S-labeled CENP-H-I-K. Bound proteins and input were separated by SDS-PAGE, stained with CBB (left), and analyzed with a phosphorimager (right). CENP-H and -K co-migrate. The asterisk indicates a CENP-K fragment. Broken lines indicate that intervening lanes have been spliced out.

    Journal: The Journal of Cell Biology

    Article Title: Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores

    doi: 10.1083/jcb.201407074

    Figure Lengend Snippet: CENP-T promotes KMN kinetochore targeting through CENP-H-I-K. (A) HeLa Tet-On cells were mock transfected or transfected with siCENP-H, treated with thymidine for 14 h, released into nocodazole-containing medium for 12 h, and treated with or without ZM for 2 h. Their mitotic index was determined by flow cytometry. Means and SDs (error bars) are shown ( n = 3 independent experiments). (B) HeLa cells were transfected with the indicated plasmids and siRNAs (siCC, siCENP-C; siCC+CH, siCENP-C+siCENP-H), and treated with nocodazole. Their mitotic index (mean ± SD [error bars], n = 3) was quantified by flow cytometry. (C and D) Nocodazole-treated mitotic HeLa cells transfected with siCENP-H were further incubated with MG132 (NM) or with both MG132 and ZM (NM+Z), and stained with the indicated antibodies and DAPI. Boxed regions of merged images were magnified and shown in the rightmost column. The relative kinetochore intensities (mean ± SD, n = 400) in certain channels were quantified and shown. Bars, 5 µm (1 µm for magnified images). (E) Recombinant Ndc80C was preincubated with or without recombinant Mis12C, immobilized on beads, and incubated with 35 S-labeled CENP-H-I-K. Bound proteins and input were separated by SDS-PAGE, stained with CBB (left), and analyzed with a phosphorimager (right). CENP-H and -K co-migrate. The asterisk indicates a CENP-K fragment. Broken lines indicate that intervening lanes have been spliced out.

    Article Snippet: HeLa Tet-On cells stably expressing Dsn1-EGFP or mCherry-CENP-T were maintained with the growth medium containing 150 µg/ml hygromycin (Takara Bio Inc.).

    Techniques: Transfection, Flow Cytometry, Cytometry, Incubation, Staining, Recombinant, Labeling, SDS Page

    Endogenous mitofusins locate on the mitochondrion-peroxisome contacting sites. a Immunofluorescence images of endogenous MFN2. Confocal microscopy analysis of HeLa cells expressing COX4-EGFP (mitochondria), immunostained for MFN2 (Alexa Fluor 555) and peroxisomal protein ABCD3 (Alexa Fluor 647). Scale bars: 5 μm. b The zoomed-in images of the white box in ( a ). White arrows point the places where ABCD3 overlaps with MFN. Scale bars: 1 μm. c Histograms display measured fluorescence intensity along the white line in the merge panels in b , with the cyan line represents mitochondria, the magenta line represents endogenous MFN2, and the red line represents peroxisome. d – f Immunofluorescence images and analysis of endogenous MFN1. Similar experiments were performed as in ( a – c ).

    Journal: Communications Biology

    Article Title: The MFN1 and MFN2 mitofusins promote clustering between mitochondria and peroxisomes

    doi: 10.1038/s42003-022-03377-x

    Figure Lengend Snippet: Endogenous mitofusins locate on the mitochondrion-peroxisome contacting sites. a Immunofluorescence images of endogenous MFN2. Confocal microscopy analysis of HeLa cells expressing COX4-EGFP (mitochondria), immunostained for MFN2 (Alexa Fluor 555) and peroxisomal protein ABCD3 (Alexa Fluor 647). Scale bars: 5 μm. b The zoomed-in images of the white box in ( a ). White arrows point the places where ABCD3 overlaps with MFN. Scale bars: 1 μm. c Histograms display measured fluorescence intensity along the white line in the merge panels in b , with the cyan line represents mitochondria, the magenta line represents endogenous MFN2, and the red line represents peroxisome. d – f Immunofluorescence images and analysis of endogenous MFN1. Similar experiments were performed as in ( a – c ).

    Article Snippet: BCCP-PupE-IRES-BFP was cloned into the expression plasmid of the Tet-On 3G inducible expression system (Clontech cat#: 631168).

    Techniques: Immunofluorescence, Confocal Microscopy, Expressing, Fluorescence

    Exogenous expression of MFNs enhance the PerMit Venus reporter signal. a Schematic for the constructs of Cyto-V(N), Mito-V(N), and Po-V(C). Cyto-V(N), HA tag fused to the N terminus of Venus with a linker composed of 4 × GGSG (indicated with blue box); Mito-V(N), Tom20 fused to the N terminus Venus with a HA tag and two linkers as indicated; Po-V(C), Myc tag and PEX26(residues 237-305) fused to the C terminus of Venus (residues 155-238). b Immuno-blots for Cyto-V(N), Mito-V(N), and Po-V(C). c Immunofluorescence images of Cyto-V(N), Mito-V(N), and Po-V(C) co-stained with mitochondrial COX4 and peroxisomal ABCD3 in HeLa cells. Scale bars, 5 μm. d Immunofluorescence images of Venus co-stained with mitochondrial COX4 and peroxisomal ABCD3 in PerMit Venus and control cells. Scale bars, 5 μm. e Immunofluorescence images of PerMit Venus cells with exogenously expressed MFNs. PerMit Venus cells were transfected with empty vector, MFN1-FLAG, or MFN2-FLAG plasmids for 36 h. FLAG (Alexa Fluor 568) and peroxisomal membrane protein PEX14 (Alexa Fluor 647) were immunostained. Scale bars, 5 μm. f Integrated density of ( e ), Vector, n = 67; MFN1-FLAG, n = 73; MFN2-FLAG, n = 72. *** p

    Journal: Communications Biology

    Article Title: The MFN1 and MFN2 mitofusins promote clustering between mitochondria and peroxisomes

    doi: 10.1038/s42003-022-03377-x

    Figure Lengend Snippet: Exogenous expression of MFNs enhance the PerMit Venus reporter signal. a Schematic for the constructs of Cyto-V(N), Mito-V(N), and Po-V(C). Cyto-V(N), HA tag fused to the N terminus of Venus with a linker composed of 4 × GGSG (indicated with blue box); Mito-V(N), Tom20 fused to the N terminus Venus with a HA tag and two linkers as indicated; Po-V(C), Myc tag and PEX26(residues 237-305) fused to the C terminus of Venus (residues 155-238). b Immuno-blots for Cyto-V(N), Mito-V(N), and Po-V(C). c Immunofluorescence images of Cyto-V(N), Mito-V(N), and Po-V(C) co-stained with mitochondrial COX4 and peroxisomal ABCD3 in HeLa cells. Scale bars, 5 μm. d Immunofluorescence images of Venus co-stained with mitochondrial COX4 and peroxisomal ABCD3 in PerMit Venus and control cells. Scale bars, 5 μm. e Immunofluorescence images of PerMit Venus cells with exogenously expressed MFNs. PerMit Venus cells were transfected with empty vector, MFN1-FLAG, or MFN2-FLAG plasmids for 36 h. FLAG (Alexa Fluor 568) and peroxisomal membrane protein PEX14 (Alexa Fluor 647) were immunostained. Scale bars, 5 μm. f Integrated density of ( e ), Vector, n = 67; MFN1-FLAG, n = 73; MFN2-FLAG, n = 72. *** p

    Article Snippet: BCCP-PupE-IRES-BFP was cloned into the expression plasmid of the Tet-On 3G inducible expression system (Clontech cat#: 631168).

    Techniques: Expressing, Construct, Western Blot, Immunofluorescence, Staining, Transfection, Plasmid Preparation

    Exogenous expression of MFN induces peroxisome/mitochondrion clustering. Immunofluorescence images of overexpressed MFN-EGFP. HeLa cells were transfected with free EGFP ( a ) or EGFP fused MFN ( b ) plasmids for 36 h. Peroxisomal matrix protein catalase (Alexa Fluor 555) and peroxisomal membrane protein ABCD3 (Alexa Fluor 647) were immune-stained. Scale bars, 5 μm. c Peroxisomal membrane protein PEX14 (Alexa Fluor 555) and outer mitochondrial membrane protein Tom20 (Alexa Fluor 647) were immunostained with or without exogenously expressed MFNs. Scale bars, 5 μm. d Immuno-blots of overexpressed MFN1-EGFP and MFN2-EGFP. 1.5 µg plasmids were transfected into HeLa cells in one well in a six-well cell culture plate for 36 h and immunoblotted with indicated antibodies. e Immunofluorescence images of overexpressed MFN2-EGFP and other organelle markers. HeLa cells were transfected with MFN2-EGFP and immunostained for peroxisomal membrane protein ABCD3 (Alexa Fluor 647) and other organelle markers (Alexa Fluor 555): calnexin (endoplasmic reticulum), EEA1 (early endosome), GM130 (Golgi), and LAMP1 (lysosome). Scale bars, 5 μm. f Immunofluorescence images of overexpressed MFN1-EGFP and other organelle markers stained the same as in ( c ).

    Journal: Communications Biology

    Article Title: The MFN1 and MFN2 mitofusins promote clustering between mitochondria and peroxisomes

    doi: 10.1038/s42003-022-03377-x

    Figure Lengend Snippet: Exogenous expression of MFN induces peroxisome/mitochondrion clustering. Immunofluorescence images of overexpressed MFN-EGFP. HeLa cells were transfected with free EGFP ( a ) or EGFP fused MFN ( b ) plasmids for 36 h. Peroxisomal matrix protein catalase (Alexa Fluor 555) and peroxisomal membrane protein ABCD3 (Alexa Fluor 647) were immune-stained. Scale bars, 5 μm. c Peroxisomal membrane protein PEX14 (Alexa Fluor 555) and outer mitochondrial membrane protein Tom20 (Alexa Fluor 647) were immunostained with or without exogenously expressed MFNs. Scale bars, 5 μm. d Immuno-blots of overexpressed MFN1-EGFP and MFN2-EGFP. 1.5 µg plasmids were transfected into HeLa cells in one well in a six-well cell culture plate for 36 h and immunoblotted with indicated antibodies. e Immunofluorescence images of overexpressed MFN2-EGFP and other organelle markers. HeLa cells were transfected with MFN2-EGFP and immunostained for peroxisomal membrane protein ABCD3 (Alexa Fluor 647) and other organelle markers (Alexa Fluor 555): calnexin (endoplasmic reticulum), EEA1 (early endosome), GM130 (Golgi), and LAMP1 (lysosome). Scale bars, 5 μm. f Immunofluorescence images of overexpressed MFN1-EGFP and other organelle markers stained the same as in ( c ).

    Article Snippet: BCCP-PupE-IRES-BFP was cloned into the expression plasmid of the Tet-On 3G inducible expression system (Clontech cat#: 631168).

    Techniques: Expressing, Immunofluorescence, Transfection, Staining, Western Blot, Cell Culture