coding sequences of human ubxn1 (uniprot identifier q04323-1) and faf2 (uniprot identifier q96cs3-1)  (GenScript corporation)

Journal: eLife

Article Title: Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase

doi: 10.7554/eLife.76763

Figure Lengend Snippet: ( A ) Domain organisation of the indicated UBX proteins. UBA = ‘ UB iquitin- A ssociated’ domain that binds ubiquitin; UBX = ‘ UB iquitin regulatory X ’ domain that binds p97; UBL = ‘ UB iquitin- L ike’ domain that binds ubiquitin and NEDD8; UAS = domain of unknown function in FAF1/FAF2/UBXN7. ( B ) Purified proteins – the membrane anchoring domain of FAF2 was deleted in FAF2 ∆M to facilitate expression of a soluble protein. ( C–E ) CMG disassembly reactions in the presence of the indicated factors, performed as described in — . See also – . Figure 4—source data 1. Source data for .

Article Snippet: The coding sequences of human UBXN1 (Uniprot identifier Q04323-1) and FAF2 (Uniprot identifier Q96CS3-1) were synthesised by GenScript Biotech.

Techniques: Ubiquitin Proteomics, Purification, Membrane, Expressing

Journal: eLife

Article Title: Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase

doi: 10.7554/eLife.76763

Figure Lengend Snippet: ( A ) Truncations of UBXN7. ( B ) Purified proteins. ( C ) CMG was ubiquitylated as in and then bound to anti-FLAG beads. Disassembly reactions were performed as above, in the presence of the indicated factors. ( D–F ) Equivalent analysis for FAF1. ( G–I ) Analogous truncations of FAF2 – ‘∆M’ indicates alleles that contain the amino terminus of the protein but lack the membrane anchoring domain. For ( A ), ( D ), and ( G ), the numbers correspond to residues in the full-length proteins. Domains were predicted using the SMART algorithm ( http://smart.embl-heidelberg.de/ ) and Alphafold . Figure 5—source data 1. Source data for .

Article Snippet: The coding sequences of human UBXN1 (Uniprot identifier Q04323-1) and FAF2 (Uniprot identifier Q96CS3-1) were synthesised by GenScript Biotech.

Techniques: Purification, Membrane

Journal: eLife

Article Title: Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase

doi: 10.7554/eLife.76763

Figure Lengend Snippet: ( A ) Human and mouse orthologues of FAF1 were aligned with Clustal Omega software and the output viewed with MView . ( B, C ) Similar analysis for FAF2 and UBXN7.

Article Snippet: The coding sequences of human UBXN1 (Uniprot identifier Q04323-1) and FAF2 (Uniprot identifier Q96CS3-1) were synthesised by GenScript Biotech.

Techniques: Software

Journal: eLife

Article Title: Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase

doi: 10.7554/eLife.76763

Figure Lengend Snippet: ( A–C ) Loci and corresponding guide RNAs (gRNAs) that were used to target the D10A ‘nickase’ mutant of Cas9 to the Faf1 , Ubxn7 , and Faf2 loci in mouse ES cells. See Materials and methods for further details. PAM = ‘Protospacer Adjacent Motif’ that takes the form ‘NGG’ and is essential for cutting by Cas9. ( D ) Immunoblot analysis with the indicated antibodies to monitor single and double deletion of the Faf1 and Ubxn7 genes. The right panel shows total protein stained with Ponceau S. ( E ) Analogous immunoblot analysis of the indicated strains, including deletion of the UBX domain of FAF2. ( F ) Doubling time of mES cells with the indicated genotypes was monitored as described in Materials and methods. The histogram indicates the mean values from three independent experiments, together with the associated standard deviations. Figure 6—figure supplement 3—source data 1. Source data for .

Article Snippet: The coding sequences of human UBXN1 (Uniprot identifier Q04323-1) and FAF2 (Uniprot identifier Q96CS3-1) were synthesised by GenScript Biotech.

Techniques: Mutagenesis, Western Blot, Staining

Journal: bioRxiv

Article Title: Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase

doi: 10.1101/2022.01.13.476277

Figure Lengend Snippet: UBXN7, FAF1 and FAF2 reduce the ubiquitin threshold of p97-UFD1-NPL4. ( A ) Domain organisation of the indicated UBX proteins. UBA = ‘ UB iquitin- A ssociated’ domain that binds ubiquitin; UBX = ‘ UB iquitin regulatory X ’ domain that binds p97; UBL = ‘UBiquitin- L ike’ domain that binds ubiquitin and NEDD8; UAS = domain of unknown function in FAF1 / FAF2 / UBXN7. ( B ) Purified proteins – the transmembrane domain of FAF2 was deleted in FAF2 ΔT to facilitate expression of a soluble protein. ( C - E) CMG disassembly reactions in the presence of the indicated factors, performed as described above for - . See also - .

Article Snippet: The coding sequences of human UBXN1 (Uniprot identifier Q04323-1) and FAF2 (Uniprot identifier Q96CS3-1) were synthesized by GenScript Biotech.

Techniques: Ubiquitin Proteomics, Purification, Expressing

Journal: bioRxiv

Article Title: Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase

doi: 10.1101/2022.01.13.476277

Figure Lengend Snippet: Mapping domains of UBXN7, FAF1 and FAF2 that stimulate the unfoldase activity of p97-UFD1-NPL4. ( A ) Truncations of UBXN7. ( B ) Purified proteins. ( C ) CMG disassembly reactions in the presence of the indicated factors, performed as described above. ( D - F ) Equivalent analysis for FAF1. ( G - I ) Analogous truncations of FAF2 – ‘ΔT’ indicates alleles that contain the amino terminus of the protein but lack the transmembrane domain. For (A), (D) and (G), the numbers correspond to residues in the full-length proteins. Domains were predicted using the SMART algorithm ( http://smart.embl-heidelberg.de/ ) and Alphafold .

Article Snippet: The coding sequences of human UBXN1 (Uniprot identifier Q04323-1) and FAF2 (Uniprot identifier Q96CS3-1) were synthesized by GenScript Biotech.

Techniques: Activity Assay, Purification

Journal: bioRxiv

Article Title: Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase

doi: 10.1101/2022.01.13.476277

Figure Lengend Snippet: Sequence alignment of human and mouse orthologues of FAF1, FAF2 and UBXN7. ( A ) Human and mouse orthologues of FAF1 were aligned with Clustal Omega software and the output viewed with MView . ( B - C ) Similar analysis for FAF2 and UBXN7.

Article Snippet: The coding sequences of human UBXN1 (Uniprot identifier Q04323-1) and FAF2 (Uniprot identifier Q96CS3-1) were synthesized by GenScript Biotech.

Techniques: Sequencing, Software

Journal: bioRxiv

Article Title: Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase

doi: 10.1101/2022.01.13.476277

Figure Lengend Snippet: Deletion of the FAF1 , UBXN7 and FAF2 genes by CRISPR-Cas9. ( A - C ) Loci and corresponding guide RNAs (gRNAs) that were used to target the D10A ‘nickase’ mutant of Cas9 to the FAF1 , UBXN7 and FAF2 loci in mouse ES cells. See Materials and Methods for further details. PAM = ‘Protospacer Adjacent Motif’ that takes the form ‘NGG’ and is essential for cutting by Cas9. ( D ) Immunoblot analysis with the indicated antibodies to monitor deletion of the FAF1 , FAF2 and UBNX7 genes. The right panels show total protein stained with Ponceau S. ( E ) Doubling time of mES cells with the indicated genotypes was monitored as described in Materials and Methods. The histogram indicates the mean values from three biological replicates, together with the associated standard deviations.

Article Snippet: The coding sequences of human UBXN1 (Uniprot identifier Q04323-1) and FAF2 (Uniprot identifier Q96CS3-1) were synthesized by GenScript Biotech.

Techniques: CRISPR, Mutagenesis, Western Blot, Staining