Journal:

Article Title: A Family of Mammalian E3 Ubiquitin Ligases That Contain the UBR Box Motif and Recognize N-Degrons

doi: 10.1128/MCB.25.16.7120-7136.2005

Figure Lengend Snippet: Identification of mouse N-recognins. (A) Peptide-pulldown assay using testis extracts and bead-conjugated peptides. Captured proteins were separated and visualized using silver staining. The identities of four major bands that are specifically and reproducibly captured by Phe- and/or Arg-peptide beads were determined using peptide mass fingerprinting. (B) UBR1, UBR2, and UBR4 bound from testis extract were eluted from Phe-peptide beads by the Phe-Ala dipeptide but not by Gly-Ala. (C) The amount of UBR4 bound by Phe-peptide beads from EF cell extracts does not depend strongly on the presence of UBR1 and/or UBR2. Proteins bound by either mock or Phe-peptide beads are indicated on the left with short bars: white, UBR1; gray, UBR2; and black, UBR4. (D) Testis proteins bound to bead-conjugated peptides bearing different N-terminal amino acids were immunoblotted with antibodies against UBR1, UBR2, UBR4, and UBR5. Mock, beads without peptide conjugation. (E) Semipurification of endogenous UBR1/UBR2 from an EF cytoplasmic extract using a peptide-pulldown assay. The precipitates/Phe-peptide beads complex prepared from cytoplasmic EF extracts were separated on SDS-PAGE and transferred onto a polyvinylidene difluoride membrane, followed by staining with Coomassie brilliant blue R-250 (Bio-Rad). Anti-UBR1 and -UBR2 immunoblotting confirmed enrichment of UBR1 and UBR2 (data not shown). (F) Northern blot analysis of UBR4 and β-actin using different mouse adult tissues. Total RNA (20 μg) was loaded in each lane. Ethidium bromide-stained ribosomal RNAs are shown as a loading control (bottom panel).

Article Snippet: Chicken polyclonal antibodies to mouse UBR4 were produced by Gallus Immunotech Inc. (Ontario, Canada) against synthetic peptides CSFEKYDEDHSGDDK (residues 4615 to 4629; antibody UBR4-119R) and DLDGEDEKDKGALDC (residues 2982 to 2995, plus an additional C-terminal Cys; antibody UBR4-244).

Techniques: Silver Staining, Peptide Mass Fingerprinting, Conjugation Assay, SDS Page, Staining, Western Blot, Northern Blot

Journal:

Article Title: A Family of Mammalian E3 Ubiquitin Ligases That Contain the UBR Box Motif and Recognize N-Degrons

doi: 10.1128/MCB.25.16.7120-7136.2005

Figure Lengend Snippet: (A) Sequence alignment of the UBR box motifs. Conserved Cys and His residues are highlighted (cyan). Indicated by orange highlight is the Cys residue of Arabidopsis BIG, whose missense mutation perturbs auxin transport (24). Indicated by pink are the residues of S. cerevisiae UBR1 essential for degradation of type 1 N-end rule substrates (A. Webster, M. Ghislain, and A. Varshavsky, unpublished data). Note that different organisms contain different sets of UBR box proteins. Prefixes: m, Mus musculus; d, Drosophila melanogaster; a, Arabidopsis thaliana; sc, Saccharomyces cerevisiae; sp, Schizosaccharomyces pombe, c, Caenorhabditis elegans. Protein sequences listed are m-UBR1 (NP_033487), m-UBR2 (AAQ17202), m-UBR3 (T. Tasaki, Y. T. Kwon, and A. Varshavsky, unpublished data), m-UBR4 (this study), m-UBR5 (AAT28194), m-UBR6 (AAH55343), m-UBR7 (BAC40212), d-UBR1 (NP_573184), d-UBR3 (NP_572426), d-UBR4 (NP_476986), d-UBR5 (P51592), d-UBR6 (AAF54459), d-UBR7 (AAF53607), c-UBR1 (AAB42328), c-UBR3 (AAO38656), c-UBR4 (AAD47122), c-UBR5 (NP_492389), c-UBR6 (AAC48052), c-UBR7 (CAA99920), a-UBR1 (NP_195851), a-UBR4 (NP_186875), a-UBR7 (T08905), sc-UBR1 (P19812), sc-UBR2 (NP_013124), sp-UBR1 (CAB10108), sp-UBR3 (O60152), and sp-UBR7 (Q09329). (B) Locations of the UBR boxes and domains characteristic, in particular, of E3 Ub ligases in the mouse UBR1 to UBR7 proteins. UBR, UBR box; RING, RING finger; CRD, cysteine-rich domain; HECT, HECT domain; PHD, plant homeodomain. (C) Phylogenetic relationships of UBR box proteins. The relative evolutional distances between each UBR box sequence were calculated by a neighbor-joining method. The tree is unrooted and the length of each branch is proportional.

Article Snippet: Chicken polyclonal antibodies to mouse UBR4 were produced by Gallus Immunotech Inc. (Ontario, Canada) against synthetic peptides CSFEKYDEDHSGDDK (residues 4615 to 4629; antibody UBR4-119R) and DLDGEDEKDKGALDC (residues 2982 to 2995, plus an additional C-terminal Cys; antibody UBR4-244).

Techniques: Sequencing, Mutagenesis

Journal:

Article Title: A Family of Mammalian E3 Ubiquitin Ligases That Contain the UBR Box Motif and Recognize N-Degrons

doi: 10.1128/MCB.25.16.7120-7136.2005

Figure Lengend Snippet: Analysis of the in vivo degradation of Sindbis virus RNA polymerases bearing N-terminal Tyr in wild-type, UBR4RNAi, UBR1−/− UBR2−/− UBR4RNAi, and UBR1−/− UBR2−/− LucRNAi stable cell lines. (A) Immunoblot analysis of UBR4 in UBR4RNAi, UBR1−/− UBR2−/− UBR4RNAi, and UBR1−/− UBR2−/− LucRNAi stable cell lines. Wild-type (wt) and UBR−/− UBR2−/− EFs stably expressing UBR4 short-hairpin RNA (shRNA) were analyzed by immunoblot using anti-UBR4 antibody. Cells were transduced by recombinant retroviruses with four different short-hairpin RNA sequences (A, B, C, and D) for UBR4 RNAi or firefly luciferase (Luc) and subsequently selected for puromycin resistance. After drug selection, a pool of colonies was lysed, and UBR4 knockdown efficiency was determined using 5% SDS-PAGE and immunoblotting. A nonspecific band is indicated by the asterisk as a loading control. Independent immunoblot analysis with isolated colonies gave largely similar results (data not shown). (B) Pulse-chase analysis of X-nsP4, where X is Met (stabilizing), Arg (type 1 N-degron), or Tyr (type 2 N-degron), in cells deficient in UBR proteins, singly or in combination. See Fig. ​Fig.1F1F for details. (C) Quantitation of the patterns shown in B using PhosphorImager.

Article Snippet: Chicken polyclonal antibodies to mouse UBR4 were produced by Gallus Immunotech Inc. (Ontario, Canada) against synthetic peptides CSFEKYDEDHSGDDK (residues 4615 to 4629; antibody UBR4-119R) and DLDGEDEKDKGALDC (residues 2982 to 2995, plus an additional C-terminal Cys; antibody UBR4-244).

Techniques: In Vivo, Stable Transfection, Western Blot, Expressing, shRNA, Recombinant, Luciferase, Selection, SDS Page, Isolation, Pulse Chase, Quantitation Assay

Journal:

Article Title: A Family of Mammalian E3 Ubiquitin Ligases That Contain the UBR Box Motif and Recognize N-Degrons

doi: 10.1128/MCB.25.16.7120-7136.2005

Figure Lengend Snippet: Half-lives of X-nsP4 in wild-type and mutant EF cells a

Article Snippet: Chicken polyclonal antibodies to mouse UBR4 were produced by Gallus Immunotech Inc. (Ontario, Canada) against synthetic peptides CSFEKYDEDHSGDDK (residues 4615 to 4629; antibody UBR4-119R) and DLDGEDEKDKGALDC (residues 2982 to 2995, plus an additional C-terminal Cys; antibody UBR4-244).

Techniques: Mutagenesis

Journal:

Article Title: A Family of Mammalian E3 Ubiquitin Ligases That Contain the UBR Box Motif and Recognize N-Degrons

doi: 10.1128/MCB.25.16.7120-7136.2005

Figure Lengend Snippet: Initial decay of X-nsP4 in wild-Type and mutant EF cells a

Article Snippet: Chicken polyclonal antibodies to mouse UBR4 were produced by Gallus Immunotech Inc. (Ontario, Canada) against synthetic peptides CSFEKYDEDHSGDDK (residues 4615 to 4629; antibody UBR4-119R) and DLDGEDEKDKGALDC (residues 2982 to 2995, plus an additional C-terminal Cys; antibody UBR4-244).

Techniques: Mutagenesis