ubr5 cdna  (Addgene inc)

Journal: Molecular Biology of the Cell

Article Title: The E3 ubiquitin ligase UBR5 regulates centriolar satellite stability and primary cilia

doi: 10.1091/mbc.E17-04-0248

Figure Lengend Snippet: UBR5 depletion disrupts centriolar satellite stability and primary cilia formation in hTERT-RPE1 cells. (A) Schematic describing ciliogenesis assay. (B) Depletion of UBR5 attenuates primary cilia formation in and causes dispersion of centriolar satellites in RPE1 cells. Cilium and basal body stained with antibody to glutamylated tubulin (red), satellites stained with antibody to PCM1 (green), and nucleus marked with Hoechst33258 (blue). A similar phenotype is observed for depletion of PCM1. High-powered inset fields are indicated by a dashed box. Transfections performed 72 h before imaging. Data representative of two independent experiments, with 150 cells counted per condition per experiment. Confocal imaging, bar = 10 µm. High magnification of region of interest (ROI) bar = 2 µm. (C) Quantitation of data depicted in B shows the strong penetrance of siUBR5 and siPCM1 phenotype. Blue and red bars show percentage of cells with distinct or dispersed satellites, respectively. Error bars = SEM. (D) Immunoblot showing siRNA efficacy in RPE1 cells used for staining in C. (E) Coimmunoprecipitation of UBR5 and Western blotting for potential interactions with PCM1, MIB1, and γ-tubulin (a marker of the centrosome). Transfections performed 48 h before imaging. Data representative of two experiments.

Article Snippet: Full-length UBR5 ORF was obtained from pEGFP-C1 EDD (#37190; Addgene, Cambridge, MA; Henderson et al. , 2002 ) and used to create pENTR221- UBR5 (Addgene; #81062).

Techniques: Dispersion, Staining, Transfection, Imaging, Quantitation Assay, Western Blot, Marker

Journal: Molecular Biology of the Cell

Article Title: The E3 ubiquitin ligase UBR5 regulates centriolar satellite stability and primary cilia

doi: 10.1091/mbc.E17-04-0248

Figure Lengend Snippet: Coexpression of UBR5 gene. (A) Waterfall plot showing global correlation of gene expression against UBR5 expression in NCI-60 cancer cell line panel, obtained via CellMiner ( Reinhold et al. , 2012 ). Cutoff set at Pearson correlation of 0.254. Putative ciliary proteins are indicated, with putative top tier cilia localizing proteins labeled and indicated with larger red dots; second tier cilia localizing proteins are also labeled and shown with larger gray dots. Putative cilia localizing proteins based on cilia proximity labeling data ( Mick et al. , 2015 ). (B) Structure of CSPP1 protein isoforms. Note that the extra 294 amino acid N-terminal region of CSPP-L is the binding region of the selected CSPP1 antibody. N-Degron motif shown in red. Coiled-coil domains are shown with a black box. Proline-rich domains are shown with a white box. Glutamate-rich domains are shown with a white striped box.

Article Snippet: Full-length UBR5 ORF was obtained from pEGFP-C1 EDD (#37190; Addgene, Cambridge, MA; Henderson et al. , 2002 ) and used to create pENTR221- UBR5 (Addgene; #81062).

Techniques: Gene Expression, Expressing, Labeling, Binding Assay

Journal: Molecular Biology of the Cell

Article Title: The E3 ubiquitin ligase UBR5 regulates centriolar satellite stability and primary cilia

doi: 10.1091/mbc.E17-04-0248

Figure Lengend Snippet: UBR5 binds and polyubiquitylates CSPP-L. (A) Schematic showing the structure of UBR5 protein and the position of known functional domains and amino acid changes of mutants. (B) Coimmunoprecipitation and immunoblot analysis of GFP-UBR5 (and functional mutants) transfected into HEK293T cells. GFP-UBR5 coimmunoprecipitates endogenous ubiquitylated CSPP-L. Covalent modification of CSPP-L is quantified by an integrated density plot showing relative size shift. Note the reduction of modified CSPP-L binding HECT* mutant. GFP-UBR5 and mutants are predicted to be approximately 338 kDa. Transfections performed 24 h before harvest. WCE, whole cell extract; NB, nonbound fraction (4% of input); IP, immunoprecipitation. Data are representative of two experiments. (C) Coimmunoprecipitation and immunoblot analysis of GFP-CSPP-L transfected into HEK293T cells. GFP-CSPP-L binds endogenous UBR5. Endogenous Ub is detected covalently bound to GFP-CSPP-L. The far right panel shows coimmunoprecipitation performed under denaturing conditions. GFP-CSPP-L is predicted to be approximately 170 kDa. Data are representative of two experiments.

Article Snippet: Full-length UBR5 ORF was obtained from pEGFP-C1 EDD (#37190; Addgene, Cambridge, MA; Henderson et al. , 2002 ) and used to create pENTR221- UBR5 (Addgene; #81062).

Techniques: Functional Assay, Western Blot, Transfection, Modification, Binding Assay, Mutagenesis, Immunoprecipitation

Journal: Molecular Biology of the Cell

Article Title: The E3 ubiquitin ligase UBR5 regulates centriolar satellite stability and primary cilia

doi: 10.1091/mbc.E17-04-0248

Figure Lengend Snippet: UBR5 interacts with CSPP-L at the centrosome. (A) Left to right, CSPP-L interacts with UBR5 in large foci adjacent to the nucleus (arrows). CSPP-L interacts with Ub in large foci adjacent to the nucleus (arrows). Interaction between UBR5 and Ub in HEK293T cells shows nuclear and cytoplasmic localization, with large foci of UBR5 and Ub in the nucleus. Untransfected cells show no visible signal. Nuclear marker is H2B-mCherry (magenta). High-powered inset fields are indicated by a dashed box (bar = 30 µm) and schematic of BiFC analysis for protein–protein interactions included. Data are representative of two experiments. (B) Immunoblot data showing correct production of fusion proteins in BiFC assay. Expected fusion protein sizes are V2-CSPP-L 152 kDa, V1-UBR5 329 kDa, V1-Ub 28 kDa, and V2-Ub 19 kDa. (C) CSPP-L/Ub interacting pairs colocalize with PCM1 (centriolar satellite marker) in hTERT-RPE1 cells. Bar = 20 µm. High magnification of ROI bar = 2 µm. (D) High-level expression of CSPP-L/Ub BiFC vectors shows CSPP-L and Ub interaction is confined to the centriolar satellites, despite strong detection of CSPP-L at the MTs in HEK293T cells. Region of interest (ROI) 1 shows relatively high BiFC pair expression, ROI 2 shows relatively low BiFC pair expression, and ROI 3 shows no BiFC pair expression. Bar = 10 µm. High magnification of ROI bar = 2 µm.

Article Snippet: Full-length UBR5 ORF was obtained from pEGFP-C1 EDD (#37190; Addgene, Cambridge, MA; Henderson et al. , 2002 ) and used to create pENTR221- UBR5 (Addgene; #81062).

Techniques: Marker, Protein-Protein interactions, Western Blot, Bimolecular Fluorescence Complementation Assay, Expressing

Journal: Molecular Biology of the Cell

Article Title: The E3 ubiquitin ligase UBR5 regulates centriolar satellite stability and primary cilia

doi: 10.1091/mbc.E17-04-0248

Figure Lengend Snippet: UBR5 maintains CSPP-L at the centrosome/centriolar satellites, and is required for centriolar satellite stability. (A) Depletion of UBR5 by shRNA in HEK293T cells does not decrease CSPP-L levels. shRNA induced with 1 µg/ml doxycycline 48 h before harvest. Data summary represents results from four independent experiments; n = 6 replicates per experiment. (B) Immunoprecipitation of GFP-CSPP-L coimmunoprecipitates CSPP-L–bound lysine-48 (K48) linked Ub chains and lysine-63 (K63) linked Ub chains. Transfections performed 24 h before harvest. (C) Depletion of UBR5 (but not PCM1) causes dispersion of centrosomal CSPP-L in RPE1 cells. (D) Centrosome/centriolar satellite mask. (E) Quantitation of loss of CSPP-L/PCM1 at the centrosome according to the mask described in D. At least 30 cells were scored per sample and statistical analysis performed using two-tailed t test.

Article Snippet: Full-length UBR5 ORF was obtained from pEGFP-C1 EDD (#37190; Addgene, Cambridge, MA; Henderson et al. , 2002 ) and used to create pENTR221- UBR5 (Addgene; #81062).

Techniques: shRNA, Immunoprecipitation, Transfection, Dispersion, Quantitation Assay, Two Tailed Test