paper cdna addgene 82510 pcdna3 1 mcherry  (Addgene inc)

Journal: Molecular Biology of the Cell

Article Title: Cbx2 stably associates with mitotic chromosomes via a PRC2- or PRC1-independent mechanism and is needed for recruiting PRC1 complex to mitotic chromosomes

doi: 10.1091/mbc.E14-06-1109

Figure Lengend Snippet: Mitotic chromosomal association of PRC1 fusion proteins. (A–H) Confocal fluorescence images of PRC1 fusion proteins tagged with either YFP or Cerulean expressed in PGK12.1 ES cells in various phases of mitosis. The mCherry-H2A fusion protein was used to mark mitotic chromosomes. Prometaphase (top), metaphase (middle), and anaphase/telophase (bottom). Representative images from Z -scan stacks are presented in the figures. Scale bar, 5 μm. (I) Quantitative analysis of mitotic binding fraction of PRC1 fusion proteins at mitotic chromosomes. The mitotic binding fraction is the average of individual slices of Z -scan stacks. The data represent at least 10 cells analyzed. Error bars, SD of the mean.

Article Snippet: The sequences coding Cerulean (Addgene, Cambridge, MA), YFP ( Ren et al. , 2008 ), and mCherry (Addgene) fluorescence proteins were amplified by PCR and inserted into the pTRIPZ(M) to produce vectors pTRIPZ(M)-Cerulean, pTRIPZ(M)-YFP, and pTRIPZ(M)-mCherry.

Techniques: Fluorescence, Binding Assay

Journal: Molecular Biology of the Cell

Article Title: Cbx2 stably associates with mitotic chromosomes via a PRC2- or PRC1-independent mechanism and is needed for recruiting PRC1 complex to mitotic chromosomes

doi: 10.1091/mbc.E14-06-1109

Figure Lengend Snippet: Directly recruiting PRC1 fusion proteins to mitotic chromosomes by YFP-Cbx2 but not by YFP-Cbx2 1-498 . (A) Confocal fluorescence images of Cerulean-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins coexpressed with YFP-Cbx2 and mCherry-H2A fusion proteins in Cbx2 −/− ES cells in metaphase. Scale bar, 5 μm. (B) Confocal fluorescence images of Cerulean-Ring1b fusion protein coexpressed with YFP-Cbx2 1-498 and mCherry-H2A fusion protein in Cbx2 −/− ES cells in metaphase. Scale bar, 5 μm. (C) Quantification of mitotic chromosomal association of YFP-Cbx2 and YFP-Cbx2 1-498 fusion proteins in Cbx2 −/− ES cells. The data represent an average of at least 10 cells analyzed. Error bars, SD of the mean. (D) Quantitative analysis of mitotic chromosomal association of Cerulea-Ring1b, Cerulean-Phc1, and Cerulean-Mel18 fusion proteins coexpressed with YFP-Cbx2 and of Cerulea-Ring1b coexpressed with YFP-Cbx2 1-498 in Cbx2 −/− ES cells in metaphase. The data represent an average of at least 10 cells analyzed. Error bars, SD of the mean. (E) Photobleaching FRET images of Cerulean-Ring1b interaction with YFP-Cbx2 and YFP-Cbx2 1-498 at mitotic chromosomes. The YFP-Cbx2, YFP-Cbx2 1-498 , and Cerulean-Ring1b fusion proteins as indicated above images expressed in Cbx2 −/− ES cells. Half-area of fluorescence of YFP-Cbx2 or YFP-Cbx2 1-498 fusion proteins at mitotic chromosomes was photobleached. Z -scan imaging of live cells by confocal laser microscope was performed before (top) and after (bottom) photobleaching. The arrowheads indicate the bleaching areas.

Article Snippet: The sequences coding Cerulean (Addgene, Cambridge, MA), YFP ( Ren et al. , 2008 ), and mCherry (Addgene) fluorescence proteins were amplified by PCR and inserted into the pTRIPZ(M) to produce vectors pTRIPZ(M)-Cerulean, pTRIPZ(M)-YFP, and pTRIPZ(M)-mCherry.

Techniques: Fluorescence, Imaging, Microscopy

Journal: Molecular Biology of the Cell

Article Title: Cbx2 stably associates with mitotic chromosomes via a PRC2- or PRC1-independent mechanism and is needed for recruiting PRC1 complex to mitotic chromosomes

doi: 10.1091/mbc.E14-06-1109

Figure Lengend Snippet: Analysis of structural elements of YFP-Cbx2 fusion protein required for its targeting and immobilizing. (A) Diagram of structural domains of Cbx2. The dashed rectangles indicate the region required for targeting Cbx2 to mitotic chromosomes (left) and the region required for immobilizing Cbx2 at mitotic chromosomes (right). ATH, AT-hook domain; Cbox, chromobox domain; CHD, chromodomain domain. The numbers in parentheses indicate the starting and ending of amino acid sequence. (B) Confocal images of YFP-Cbx2 and its variant fusion proteins in metaphase of HeLa cells. YFP-Cbx2 mutant and mCherry-H2A fusion proteins were stably expressed in HeLa cells. The mCherry-H2A was used to mark mitotic chromosomes. Scale bar, 5 μm. (C) FRAP curves of interphases and metaphases of YFP-Cbx2 and its variant fusion proteins expressed in HeLa cells. FRAP analysis is described in . (D) Residence time and immobile fraction of YFP-Cbx2 variant fusion proteins at interphasic and mitotic chromatins. The residence time and immobile fraction were calculated from FRAP curves by fitting a first-order kinetic model.

Article Snippet: The sequences coding Cerulean (Addgene, Cambridge, MA), YFP ( Ren et al. , 2008 ), and mCherry (Addgene) fluorescence proteins were amplified by PCR and inserted into the pTRIPZ(M) to produce vectors pTRIPZ(M)-Cerulean, pTRIPZ(M)-YFP, and pTRIPZ(M)-mCherry.

Techniques: Sequencing, Variant Assay, Mutagenesis, Stable Transfection