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egfp fib  (Addgene inc)


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    Addgene inc egfp fib
    Egfp Fib, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pegfp+c1+fibrillarin/pEGFP-C1-Fibrillarin+(Plasmid+%2326673)/bio_rxiv__64898__2026__01__05__697638-67-34-35
    Average 92 stars, based on 12 article reviews
    egfp fib - by Bioz Stars, 2026-09
    92/100 stars

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    Amplification:

    Article Title: 1,6-hexanediol rapidly immobilizes and condenses chromatin in living human cells
    Article Snippet: .. The EGFP fragment was amplified from pEGFP-C1-Fibrillarin (#26673; Addgene) ( ) using the following primers: 5′-TGGAATTCTGCAGATGCCACCATGGTGAGCAAGGGCGAGGA-3′ and 5′-CGCCGCCACCAGATCCACCTCCACCAGATCCACCTCCACCCTTGTACAGCTCGTCCATGCCG-3′. .. The amplified coilin and EGFP fragments were joined together using standard overlapping PCR and inserted into the EcoRV site of a pEF1-FRT plasmid ( ) to obtain pEF1-EGFP-coilin-FRT using In-Fusion (639650; Takara).

    Article Title: Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy.
    Article Snippet: For the construct of HaloTag-H2B (JH1348), the H2B sequence was amplified from pminiSOG-H2B-625 and substituted EGFP in pHaloTag-EGFP (addgene #86629)26 through the In-Fusion cloning method. .. For the construct of HaloTag-Fibrillarin (JH1239), the Fibrillarin sequence was amplified from pEGFP-C1-Fibrillarin (addgene #26673)27 and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method. .. NH2‐cys(S‐Trt)‐CO‐NH‐(CH2)3‐N3 (2) Fmoc-NH-cys(S-Trt)-OH, (1) (19.8 mg, 33.8 μmol) and HATU (14.1 mg, 37.2 μmol) were dissolved in dry DMF (100 μL) in a plastic screw cap tube and 3-azidopropyl-1-amine (3.7 μL, 37.2 μmol, Click Chemistry Tools) followed by DIEA (13 μL, 74.4 μmol) were added with mixing.

    Article Title: Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy
    Article Snippet: For the construct of HaloTag-H2B (JH1348), the H2B sequence was amplified from pminiSOG-H2B-6 and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method. .. For the construct of HaloTag-Fibrillarin (JH1239), the Fibrillarin sequence was amplified from pEGFP-C1-Fibrillarin (addgene #26673) and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method. .. Fmoc-NH-cys(S-Trt)-OH, (1) (19.8 mg, 33.8 μmol) and HATU (14.1 mg, 37.2 μmol) were dissolved in dry DMF (100 μL) in a plastic screw cap tube and 3-azidopropyl-1-amine (3.7 μL, 37.2 μmol, Click Chemistry Tools) followed by DIEA (13 μL, 74.4 μmol) were added with mixing.

    Article Title: Transcriptional suppression of ribosomal DNA with phase separation.
    Article Snippet: .. The EGFP fragment was amplified from pEGFP-C1-fibrillarin (#26673, Addgene) using the following primers: 5′-GTAAACTTAAGGTTAATTAACGCCACCATGGTGAGCAAGGGCGAGGAG-3′ and 5′-GGTACCGTCGACTGCAGAATTC-3′. .. The amplified RPA194-E593Q and EGFP fragments were joined together using standard overlapping PCR and inserted into the Pac I and Age I sites of pAAVS1-NDiCRISPRi (#73498, Addgene) (68) using In-Fusion.

    Article Title: Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy
    Article Snippet: For the construct of HaloTag-H2B (JH1348), the H2B sequence was amplified from pminiSOG-H2B-6 ( ) and substituted EGFP in pHaloTag-EGFP (addgene #86629)( ) through the In-Fusion cloning method. .. For the construct of HaloTag-Fibrillarin (JH1239), the Fibrillarin sequence was amplified from pEGFP-C1-Fibrillarin (addgene #26673)( ) and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method. .. Fmoc-NH-cys(S-Trt)-OH, ( 1 ) (19.8 mg, 33.8 μmol) and HATU (14.1 mg, 37.2 μmol) were dissolved in dry DMF (100 μL) in a plastic screw cap tube and 3-azidopropyl-1-amine (3.7 μL, 37.2 μmol, Click Chemistry Tools) followed by DIEA (13 μL, 74.4 μmol) were added with mixing.

    Generated:

    Article Title: Pharmacological targeting of a PWWP domain demonstrates cooperative control of NSD2 localization
    Article Snippet: For fluorescence microscopy, C-terminal GFPSpark ® -tagged NSD2 wild-type was purchased from Sino Biological. .. NSD2 point mutants were generated using mutagenic primers with Q5 ® Site-Directed Mutagenesis Kit from New England Biolabs. pEGFP-C1-Fibrillarin was a gift from Sui Huang (Addgene plasmid # 26673; RRID:Addgene_26673) . .. Cell lines were cultured according to standard aseptic mammalian tissue culture protocols in 5% CO 2 at 37 °C with regular testing for mycoplasma contamination using the MycoAlertTM Mycoplasma Detection Kit (Lonza).

    Article Title: A chemical probe targeting the PWWP domain alters NSD2 nucleolar localization.
    Article Snippet: 1Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.. 2Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.. 3EpiCypher Inc., Durham, NC, USA.

    Mutagenesis:

    Article Title: Pharmacological targeting of a PWWP domain demonstrates cooperative control of NSD2 localization
    Article Snippet: For fluorescence microscopy, C-terminal GFPSpark ® -tagged NSD2 wild-type was purchased from Sino Biological. .. NSD2 point mutants were generated using mutagenic primers with Q5 ® Site-Directed Mutagenesis Kit from New England Biolabs. pEGFP-C1-Fibrillarin was a gift from Sui Huang (Addgene plasmid # 26673; RRID:Addgene_26673) . .. Cell lines were cultured according to standard aseptic mammalian tissue culture protocols in 5% CO 2 at 37 °C with regular testing for mycoplasma contamination using the MycoAlertTM Mycoplasma Detection Kit (Lonza).

    Article Title: A chemical probe targeting the PWWP domain alters NSD2 nucleolar localization.
    Article Snippet: 1Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.. 2Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.. 3EpiCypher Inc., Durham, NC, USA.

    Plasmid Preparation:

    Article Title: Pharmacological targeting of a PWWP domain demonstrates cooperative control of NSD2 localization
    Article Snippet: For fluorescence microscopy, C-terminal GFPSpark ® -tagged NSD2 wild-type was purchased from Sino Biological. .. NSD2 point mutants were generated using mutagenic primers with Q5 ® Site-Directed Mutagenesis Kit from New England Biolabs. pEGFP-C1-Fibrillarin was a gift from Sui Huang (Addgene plasmid # 26673; RRID:Addgene_26673) . .. Cell lines were cultured according to standard aseptic mammalian tissue culture protocols in 5% CO 2 at 37 °C with regular testing for mycoplasma contamination using the MycoAlertTM Mycoplasma Detection Kit (Lonza).

    Article Title: A chemical probe targeting the PWWP domain alters NSD2 nucleolar localization.
    Article Snippet: 1Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.. 2Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.. 3EpiCypher Inc., Durham, NC, USA.

    Construct:

    Article Title: Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy.
    Article Snippet: For the construct of HaloTag-H2B (JH1348), the H2B sequence was amplified from pminiSOG-H2B-625 and substituted EGFP in pHaloTag-EGFP (addgene #86629)26 through the In-Fusion cloning method. .. For the construct of HaloTag-Fibrillarin (JH1239), the Fibrillarin sequence was amplified from pEGFP-C1-Fibrillarin (addgene #26673)27 and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method. .. NH2‐cys(S‐Trt)‐CO‐NH‐(CH2)3‐N3 (2) Fmoc-NH-cys(S-Trt)-OH, (1) (19.8 mg, 33.8 μmol) and HATU (14.1 mg, 37.2 μmol) were dissolved in dry DMF (100 μL) in a plastic screw cap tube and 3-azidopropyl-1-amine (3.7 μL, 37.2 μmol, Click Chemistry Tools) followed by DIEA (13 μL, 74.4 μmol) were added with mixing.

    Article Title: Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy
    Article Snippet: For the construct of HaloTag-H2B (JH1348), the H2B sequence was amplified from pminiSOG-H2B-6 and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method. .. For the construct of HaloTag-Fibrillarin (JH1239), the Fibrillarin sequence was amplified from pEGFP-C1-Fibrillarin (addgene #26673) and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method. .. Fmoc-NH-cys(S-Trt)-OH, (1) (19.8 mg, 33.8 μmol) and HATU (14.1 mg, 37.2 μmol) were dissolved in dry DMF (100 μL) in a plastic screw cap tube and 3-azidopropyl-1-amine (3.7 μL, 37.2 μmol, Click Chemistry Tools) followed by DIEA (13 μL, 74.4 μmol) were added with mixing.

    Article Title: Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy
    Article Snippet: For the construct of HaloTag-H2B (JH1348), the H2B sequence was amplified from pminiSOG-H2B-6 ( ) and substituted EGFP in pHaloTag-EGFP (addgene #86629)( ) through the In-Fusion cloning method. .. For the construct of HaloTag-Fibrillarin (JH1239), the Fibrillarin sequence was amplified from pEGFP-C1-Fibrillarin (addgene #26673)( ) and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method. .. Fmoc-NH-cys(S-Trt)-OH, ( 1 ) (19.8 mg, 33.8 μmol) and HATU (14.1 mg, 37.2 μmol) were dissolved in dry DMF (100 μL) in a plastic screw cap tube and 3-azidopropyl-1-amine (3.7 μL, 37.2 μmol, Click Chemistry Tools) followed by DIEA (13 μL, 74.4 μmol) were added with mixing.

    Sequencing:

    Article Title: Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy.
    Article Snippet: For the construct of HaloTag-H2B (JH1348), the H2B sequence was amplified from pminiSOG-H2B-625 and substituted EGFP in pHaloTag-EGFP (addgene #86629)26 through the In-Fusion cloning method. .. For the construct of HaloTag-Fibrillarin (JH1239), the Fibrillarin sequence was amplified from pEGFP-C1-Fibrillarin (addgene #26673)27 and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method. .. NH2‐cys(S‐Trt)‐CO‐NH‐(CH2)3‐N3 (2) Fmoc-NH-cys(S-Trt)-OH, (1) (19.8 mg, 33.8 μmol) and HATU (14.1 mg, 37.2 μmol) were dissolved in dry DMF (100 μL) in a plastic screw cap tube and 3-azidopropyl-1-amine (3.7 μL, 37.2 μmol, Click Chemistry Tools) followed by DIEA (13 μL, 74.4 μmol) were added with mixing.

    Article Title: Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy
    Article Snippet: For the construct of HaloTag-H2B (JH1348), the H2B sequence was amplified from pminiSOG-H2B-6 and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method. .. For the construct of HaloTag-Fibrillarin (JH1239), the Fibrillarin sequence was amplified from pEGFP-C1-Fibrillarin (addgene #26673) and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method. .. Fmoc-NH-cys(S-Trt)-OH, (1) (19.8 mg, 33.8 μmol) and HATU (14.1 mg, 37.2 μmol) were dissolved in dry DMF (100 μL) in a plastic screw cap tube and 3-azidopropyl-1-amine (3.7 μL, 37.2 μmol, Click Chemistry Tools) followed by DIEA (13 μL, 74.4 μmol) were added with mixing.

    Article Title: Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy
    Article Snippet: For the construct of HaloTag-H2B (JH1348), the H2B sequence was amplified from pminiSOG-H2B-6 ( ) and substituted EGFP in pHaloTag-EGFP (addgene #86629)( ) through the In-Fusion cloning method. .. For the construct of HaloTag-Fibrillarin (JH1239), the Fibrillarin sequence was amplified from pEGFP-C1-Fibrillarin (addgene #26673)( ) and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method. .. Fmoc-NH-cys(S-Trt)-OH, ( 1 ) (19.8 mg, 33.8 μmol) and HATU (14.1 mg, 37.2 μmol) were dissolved in dry DMF (100 μL) in a plastic screw cap tube and 3-azidopropyl-1-amine (3.7 μL, 37.2 μmol, Click Chemistry Tools) followed by DIEA (13 μL, 74.4 μmol) were added with mixing.

    Cloning:

    Article Title: Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy.
    Article Snippet: For the construct of HaloTag-H2B (JH1348), the H2B sequence was amplified from pminiSOG-H2B-625 and substituted EGFP in pHaloTag-EGFP (addgene #86629)26 through the In-Fusion cloning method. .. For the construct of HaloTag-Fibrillarin (JH1239), the Fibrillarin sequence was amplified from pEGFP-C1-Fibrillarin (addgene #26673)27 and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method. .. NH2‐cys(S‐Trt)‐CO‐NH‐(CH2)3‐N3 (2) Fmoc-NH-cys(S-Trt)-OH, (1) (19.8 mg, 33.8 μmol) and HATU (14.1 mg, 37.2 μmol) were dissolved in dry DMF (100 μL) in a plastic screw cap tube and 3-azidopropyl-1-amine (3.7 μL, 37.2 μmol, Click Chemistry Tools) followed by DIEA (13 μL, 74.4 μmol) were added with mixing.

    Article Title: Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy
    Article Snippet: For the construct of HaloTag-H2B (JH1348), the H2B sequence was amplified from pminiSOG-H2B-6 and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method. .. For the construct of HaloTag-Fibrillarin (JH1239), the Fibrillarin sequence was amplified from pEGFP-C1-Fibrillarin (addgene #26673) and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method. .. Fmoc-NH-cys(S-Trt)-OH, (1) (19.8 mg, 33.8 μmol) and HATU (14.1 mg, 37.2 μmol) were dissolved in dry DMF (100 μL) in a plastic screw cap tube and 3-azidopropyl-1-amine (3.7 μL, 37.2 μmol, Click Chemistry Tools) followed by DIEA (13 μL, 74.4 μmol) were added with mixing.

    Article Title: Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy
    Article Snippet: For the construct of HaloTag-H2B (JH1348), the H2B sequence was amplified from pminiSOG-H2B-6 ( ) and substituted EGFP in pHaloTag-EGFP (addgene #86629)( ) through the In-Fusion cloning method. .. For the construct of HaloTag-Fibrillarin (JH1239), the Fibrillarin sequence was amplified from pEGFP-C1-Fibrillarin (addgene #26673)( ) and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method. .. Fmoc-NH-cys(S-Trt)-OH, ( 1 ) (19.8 mg, 33.8 μmol) and HATU (14.1 mg, 37.2 μmol) were dissolved in dry DMF (100 μL) in a plastic screw cap tube and 3-azidopropyl-1-amine (3.7 μL, 37.2 μmol, Click Chemistry Tools) followed by DIEA (13 μL, 74.4 μmol) were added with mixing.



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    Figure 6. CLEM of <t>Fibrillarin</t> binding to RNA using PROMPT. (a) Confocal micrographs of U2OS cells transfected with or without the HaloTag-Fibrillarin construct, pretreated overnight with or without 1 mM 5EU, and processed with the PROMPT procedure. (b) Quantification of the mean JF570-PROMPT fluorescence signal per nucleus in cells treated as in 6A. Error bars are box-and-whiskers plots containing the mean (X), quartiles (box), and minimum and maximum observations (whiskers). (c) DAB photooxidation by JF570- PROMPT after the PROMPT procedure in U2OS cell transfected with HaloTag-Fibrillarin construct and treated with 1 mM 5EU. The left micrographs show the fluorescence and the brightfield images pre and post photooxidation. The image on the right shows the electron microscopy micrograph of the cells shown on the left and framed in the pink box. The asterisk points at a speck of dust on the resin section. (d) Electron microscopy micrograph of the area of interest in the black box in (c). The top image shows the original acquisition, and the bottom one is a composite image including the original TEM micrograph layered with the DAB polymers extracted by thresholding and colorized in yellow. The purple dotted lines circle the Cajal bodies. The white arrows point at 250 nm diameter granules of unknown identities. The cyan dotted line surrounds darker areas of the nucleolus. No, Nucleolus; Np, Nucleoplasm; Cb, Cajal body. (e) Electron microscopy micrograph of the area of interest in the orange box in (d). Arrowheads point at some of the electron-dense DAB polymers. The dotted white line separates the nucleolus on the right from the nucleoplasm on the left.
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    Image Search Results


    Figure 6. CLEM of Fibrillarin binding to RNA using PROMPT. (a) Confocal micrographs of U2OS cells transfected with or without the HaloTag-Fibrillarin construct, pretreated overnight with or without 1 mM 5EU, and processed with the PROMPT procedure. (b) Quantification of the mean JF570-PROMPT fluorescence signal per nucleus in cells treated as in 6A. Error bars are box-and-whiskers plots containing the mean (X), quartiles (box), and minimum and maximum observations (whiskers). (c) DAB photooxidation by JF570- PROMPT after the PROMPT procedure in U2OS cell transfected with HaloTag-Fibrillarin construct and treated with 1 mM 5EU. The left micrographs show the fluorescence and the brightfield images pre and post photooxidation. The image on the right shows the electron microscopy micrograph of the cells shown on the left and framed in the pink box. The asterisk points at a speck of dust on the resin section. (d) Electron microscopy micrograph of the area of interest in the black box in (c). The top image shows the original acquisition, and the bottom one is a composite image including the original TEM micrograph layered with the DAB polymers extracted by thresholding and colorized in yellow. The purple dotted lines circle the Cajal bodies. The white arrows point at 250 nm diameter granules of unknown identities. The cyan dotted line surrounds darker areas of the nucleolus. No, Nucleolus; Np, Nucleoplasm; Cb, Cajal body. (e) Electron microscopy micrograph of the area of interest in the orange box in (d). Arrowheads point at some of the electron-dense DAB polymers. The dotted white line separates the nucleolus on the right from the nucleoplasm on the left.

    Journal: Scientific reports

    Article Title: Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy.

    doi: 10.1038/s41598-023-45413-8

    Figure Lengend Snippet: Figure 6. CLEM of Fibrillarin binding to RNA using PROMPT. (a) Confocal micrographs of U2OS cells transfected with or without the HaloTag-Fibrillarin construct, pretreated overnight with or without 1 mM 5EU, and processed with the PROMPT procedure. (b) Quantification of the mean JF570-PROMPT fluorescence signal per nucleus in cells treated as in 6A. Error bars are box-and-whiskers plots containing the mean (X), quartiles (box), and minimum and maximum observations (whiskers). (c) DAB photooxidation by JF570- PROMPT after the PROMPT procedure in U2OS cell transfected with HaloTag-Fibrillarin construct and treated with 1 mM 5EU. The left micrographs show the fluorescence and the brightfield images pre and post photooxidation. The image on the right shows the electron microscopy micrograph of the cells shown on the left and framed in the pink box. The asterisk points at a speck of dust on the resin section. (d) Electron microscopy micrograph of the area of interest in the black box in (c). The top image shows the original acquisition, and the bottom one is a composite image including the original TEM micrograph layered with the DAB polymers extracted by thresholding and colorized in yellow. The purple dotted lines circle the Cajal bodies. The white arrows point at 250 nm diameter granules of unknown identities. The cyan dotted line surrounds darker areas of the nucleolus. No, Nucleolus; Np, Nucleoplasm; Cb, Cajal body. (e) Electron microscopy micrograph of the area of interest in the orange box in (d). Arrowheads point at some of the electron-dense DAB polymers. The dotted white line separates the nucleolus on the right from the nucleoplasm on the left.

    Article Snippet: For the construct of HaloTag-Fibrillarin (JH1239), the Fibrillarin sequence was amplified from pEGFP-C1-Fibrillarin (addgene #26673)27 and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method.

    Techniques: Binding Assay, Transfection, Construct, Fluorescence, Electron Microscopy

    Figure 7. Electron Tomography of Fibrillarin/RNA PROMPT. (a) Confocal micrographs of U2OS cells transfected with the HaloTag-Fibrillarin construct, pretreated overnight with 1 mM 5EU, and processed with the PROMPT procedure. Brightfield images are acquired before and after pho-oxidation (PO) of DAB by JF570- PROMPT. The lowest image shows the corresponding photo-oxidized cell by transmission electron microscopy. The orange box delineates the area acquired by electron tomography in (b). (b) Both panels display a minimum projection of 8 consecutive electron tomogram slices from the cell treated and photo-oxidized in 7A. In the lowest image, the most electron dense areas have been segmented by thresholding and colorized in orange. No, Nucleolus; Cb, Cajal Body.

    Journal: Scientific reports

    Article Title: Proximal Molecular Probe Transfer (PROMPT), a new approach for identifying sites of protein/nucleic acid interaction in cells by correlated light and electron microscopy.

    doi: 10.1038/s41598-023-45413-8

    Figure Lengend Snippet: Figure 7. Electron Tomography of Fibrillarin/RNA PROMPT. (a) Confocal micrographs of U2OS cells transfected with the HaloTag-Fibrillarin construct, pretreated overnight with 1 mM 5EU, and processed with the PROMPT procedure. Brightfield images are acquired before and after pho-oxidation (PO) of DAB by JF570- PROMPT. The lowest image shows the corresponding photo-oxidized cell by transmission electron microscopy. The orange box delineates the area acquired by electron tomography in (b). (b) Both panels display a minimum projection of 8 consecutive electron tomogram slices from the cell treated and photo-oxidized in 7A. In the lowest image, the most electron dense areas have been segmented by thresholding and colorized in orange. No, Nucleolus; Cb, Cajal Body.

    Article Snippet: For the construct of HaloTag-Fibrillarin (JH1239), the Fibrillarin sequence was amplified from pEGFP-C1-Fibrillarin (addgene #26673)27 and substituted EGFP in pHaloTag-EGFP (addgene #86629) through the In-Fusion cloning method.

    Techniques: Tomography, Transfection, Construct, Transmission Assay, Electron Microscopy