Journal: Molecular Neurodegeneration

Article Title: VCP regulates early tau seed amplification via specific cofactors

doi: 10.1186/s13024-024-00783-z

Figure Lengend Snippet: List of VCP cofactors and their proposed functions

Article Snippet: FAF2 (Human)—3 unique 27mer siRNA duplexes—2 nmol each , OriGene , SR308083.

Techniques: Membrane

Journal: Molecular Neurodegeneration

Article Title: VCP regulates early tau seed amplification via specific cofactors

doi: 10.1186/s13024-024-00783-z

Figure Lengend Snippet: VCP cofactors differentially regulate tau seeding. VCP cofactors were either knocked out via CRISPR/Cas9 ( A-D ) or knocked down via siRNA ( E–G ) in v2L biosensors prior to exposure to increasing amounts of tau fibrils. A Knockout of FAF2 increased tau seeding whereas knockout of B ATXN3, C NSFL1C, and D UBE4B reduced tau seeding. P values: FAF2 (*** 0.0001, **** < 0.0001); ATXN3 (**** < 0.0001); NSFL1C (*** 0.0002); UBE4B (**** < 0.0001). E Knockdown of NGLY1, F NPLOC4, and G OTUB1, decreased tau seeding. P values: NGLY1(**** < 0.0001); NPLOC4 (**** < 0.0001); OTUB1 (*** 0.0004, **** < 0.0001, *** 0.0001). Graphs are representative of n = 3 independent experiments, with each data point derived from technical triplicate. Error bars represent S.D. Some error bars are too small to be visible. H Cofactor KO did not affect tau uptake. P values: ns = 0.9819, 0.9988, 0.9956, 0.9928, in order of bars on the graph. I Cofactor KD did not affect tau uptake. P values: ns = 0.9795, 0.1856, 0.3928, in order of bars on the graph. Error bars represent S.E.M ( n = 3). One-Way ANOVA with a 95% confidence interval

Article Snippet: FAF2 (Human)—3 unique 27mer siRNA duplexes—2 nmol each , OriGene , SR308083.

Techniques: CRISPR, Knock-Out, Knockdown, Derivative Assay

Journal: Molecular Neurodegeneration

Article Title: VCP regulates early tau seed amplification via specific cofactors

doi: 10.1186/s13024-024-00783-z

Figure Lengend Snippet: List of Reagents

Article Snippet: FAF2 (Human)—3 unique 27mer siRNA duplexes—2 nmol each , OriGene , SR308083.

Techniques: Protease Inhibitor, Cell Culture, Transfection, Magnetic Beads, Sequencing, Modification

Journal: Molecular Neurodegeneration

Article Title: VCP regulates early tau seed amplification via specific cofactors

doi: 10.1186/s13024-024-00783-z

Figure Lengend Snippet: List of Reagents

Article Snippet: Anti-FAF2 Rabbit Polyclonal Antibody, Size = 150 μL , Fisher Scientific , 16,251–1-AP.

Techniques: Protease Inhibitor, Cell Culture, Transfection, Magnetic Beads, Sequencing, Modification

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

Journal: PLoS ONE

Article Title: Chlamydia trachomatis Infection Leads to Defined Alterations to the Lipid Droplet Proteome in Epithelial Cells

doi: 10.1371/journal.pone.0124630

Figure Lengend Snippet: List of proteins represented abundantly in LD extracts from uninfected and C . trachomatis -infected HeLa cells.

Article Snippet: Antibodies used were: ACSL3 (rabbit polyclonal, Abcam), ACSL4 (rabbit polyclonal, ProteinTech Group), Aup-1 (rabbit polyclonal, ProteinTech Group), Calreticulin (rabbit polyclonal, Stressgen), FAF2 (mouse polyclonal, Abnova), GAPDH (rabbit polyclonal, Abcam), Integrinα2 (mouse monoclonal, BD Biosciences), NSDHL (rabbit polyclonal, Abcam), PLIN2 (mouse monoclonal, ProGen Biotechnik), PLIN3 (rabbit polyclonal, ProteinTech Group), PLPL2 (rabbit polyclonal, Abcam), Rab1 (rabbit polyclonal, Santa Cruz Biotechnology), Rab11 (mouse monoclonal, BD Biosciences), RpoB (rabbit polyclonal, kindly provided by M. Tan, UC Irvine), TRAPα (mouse monoclonal, Abcam).

Techniques: Infection