Journal: Current biology : CB

Article Title: MULTIPLE ISOFORMS OF NESPRIN1 ARE INTEGRAL COMPONENTS OF CILIARY ROOTLETS

doi: 10.1016/j.cub.2017.05.066

Figure Lengend Snippet: A. Nesprin1 and Myc colabelling of NIH3t3 cells transfected with Myc-root. Note the NE aggregation and centrosome recruitment of Nesprin1 by Myc-root filaments. See also Figure S2A,B. B, C, D. Same colabelling experiment with antibodies against Nesprin2 (B), Sun1 (C) or Sun2 (D) at the NE. Note that whereas the NE localization of Nesprin2 and Sun1 is not affected by Myc-root filaments, Sun2 aggregates into NE Myc-root/Nesprin1 filaments. See also Figures S2D–G. E. Model of Sun2/Nesprin1 LINC complexes that dock perinuclear rootletin filaments at the nuclear surface of rods and of NIH3t3 cells transfected with Myc-root. Red dots: KASH domains. F. Nesprin1 and Sun2 colabelling of the ONL of C57/Bl6 retinas. Note the discrete colocalization of Sun2 with Nesprin1 filaments (arrows). G. Myc and Nesprin1 coimmunolabeling of NIH3t3 cells transfected either with control SiHPRT (top) or with SiNes1 (bottom) SiRNAs for 36h and subsequently with Myc-root for 12h. Note the lack of rootletin filaments at the nuclear surface in the absence of Nesprin1 expression and their accumulation in the cytoplasm. See also Figure S2H. H, I: Basal views of NIH3t3 cotransfected with Myc-root and either EGFP-Nes1α (H) or EGFP-Nes1αΔKASH (I). Note that whereas EGFP-Nes1α and rootletin filaments essentially colocalize at the NE, EGFP-Nes1αΔKASH colocalizes with rootletin filaments in the cytoplasm. J: Volume to volume partition into pellets (P) and supernatant (S) of Myc-Root deletion mutants extracted with RIPA buffer from transfected NIH3t3 cells. Bottom: depiction of the coiled-coil domains of Myc-root (C1 to C6) and of each deletion mutant. K: Nesprin1 and rabbit immunoglobulins (IgG, used as a negative control) immunoprecipitations (IP) of RIPA lysates from NIH3t3 cells cotransfected with EGFP-Nes1αΔKASH and indicated Myc-Root deletion constructs. Immunoprecipitates were immunoblotted (IB) either with Myc (top panel) or with EGFP (bottom panel) antibodies to detect deletion mutants of Myc-Root and EGFP-Nes1αΔKASH, respectively. See also Figure S2I.

Article Snippet: Cell Lines NIH3t3 fibroblasts (ATCC#CRL-16580, male) were purchased from the American Type Tissue Collection through the Tissue Culture Support Center at Washington University School of Medicine.

Techniques: Transfection, Control, Expressing, Mutagenesis, Negative Control, Construct

Journal: Current biology : CB

Article Title: MULTIPLE ISOFORMS OF NESPRIN1 ARE INTEGRAL COMPONENTS OF CILIARY ROOTLETS

doi: 10.1016/j.cub.2017.05.066

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cell Lines NIH3t3 fibroblasts (ATCC#CRL-16580, male) were purchased from the American Type Tissue Collection through the Tissue Culture Support Center at Washington University School of Medicine.

Techniques: Recombinant, Electron Microscopy, Saline, Plasmid Preparation, Software

Journal: Cell Reports Methods

Article Title: Modular dual-color BiAD sensors for locus-specific readout of epigenome modifications in single cells

doi: 10.1016/j.crmeth.2024.100739

Figure Lengend Snippet: Validation of single and double reader domains as detector modules for histone PTMs (A) Representative fluorescence microscopy images of fixed NIH3T3 cells transfected with mVenus-fused HP1βCD single or double domain (2×) and the corresponding W42A binding pocket mutants. The WT 2×HP1βCD colocalizes with H3K9me3 staining at DAPI-dense heterochromatic regions, as exemplarily indicated by arrows. Dotted arrows indicate missing colocalization of the respective W42A mutants. (B) Representative fluorescence microscopy images of fixed NIH3T3 cells transfected with mVenus-fused DNMT3A PWWP single (3APWWP) or double domain (2×) and the corresponding K299E binding pocket mutants. The WT 2×3APWWP domain colocalizes with H3K36me3 immunostaining at DAPI-dense heterochromatic regions, as exemplarily indicated by arrows. Dotted arrows indicate missing colocalization of the single domain and the respective K299E mutants. (C) Representative fluorescence microscopy images of fixed HEK293 cells transfected with mVenus-fused CBX7CD single or double domain (2×) and the corresponding binding-deficient W35A mutants. 2×CBX7CD WT colocalizes with H3K27me3 and DAPI staining at the Xi, as indicated by circles. Dotted circles indicate missing colocalization of the single domain and the respective W35A mutants. In all of the panels, the images are representative of 5–15 individual cells that were analyzed. See also Figure S3 .

Article Snippet: Human embryonic kidney (HEK293; female; RRID: CVCL_0045) and NIH3T3 (male; RRID: CRL-1658) cells were obtained from DSMZ (Braunschweig, Germany) and ATCC (American Type Culture Collection), respectively.

Techniques: Biomarker Discovery, Fluorescence, Microscopy, Transfection, Binding Assay, Staining, Immunostaining

Journal: Cell Reports Methods

Article Title: Modular dual-color BiAD sensors for locus-specific readout of epigenome modifications in single cells

doi: 10.1016/j.crmeth.2024.100739

Figure Lengend Snippet:

Article Snippet: Human embryonic kidney (HEK293; female; RRID: CVCL_0045) and NIH3T3 (male; RRID: CRL-1658) cells were obtained from DSMZ (Braunschweig, Germany) and ATCC (American Type Culture Collection), respectively.

Techniques: Virus, Recombinant, Knockdown, Biomarker Discovery, Software