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

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

Journal: bioRxiv

Article Title: Unanchored ubiquitin chains promote the non-canonical inflammasome via UBXN1

doi: 10.1101/2024.10.30.621131

Figure Lengend Snippet: ( a ) Immunoprecipitation (IP) of FLAG-APEX-caspase-4 with an anti-FLAG antibody. HeLa cells were transfected with a FLAG-APEX-CASP4 and/or HA-Ub (WT) plasmid for 24 h, primed with IFN-γ for 12 h and/or transfected with LPS for 5 h. (-) stands for an empty vector plasmid. Shown are the immunoblots (IB) of indicated proteins. WCL, whole cell lysate; KO, knockout of UBXN1 . ( b ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with different combinations of FLAG-APEX-CASP4, Myc-UBXN1, Myc-UBXN3B and corresponding vector (-) for 24 h, followed by treatment with (+) / without (-) human IFN-γ and LPS as in ( a ). The endogenous Ub were examined by polyubiquitin antibodies. ( c ) IP of FLAG-APEX-caspase-4 with an anti-FLAG antibody from HeLa cells transfected with various combinations of FLAG-APEX-CASP4, Myc-UBXN1, HA-tagged WT or individual Kn-Ub (mutant) plasmids. (-) stands for an empty vector plasmid. ( d ) IP of FLAG-caspases with an anti-FLAG antibody from HeLa cells transfected with a FLAG-CASP, Myc-UBXN1 or empty vector plasmid. The red arrow heads indicate correct bands; caspase-11 shows in two bands. The endogenous total, K48, and K63 Ub were detected by specific antibodies. ( e ) IP of FLAG-caspase-4 and Myc-UBXN1 from HEK293T cells. Cells were transfected with the FLAG-CASP4, Myc-UBXN1 or both for 24 h; the cell lysates were equally split for IP with an anti-Myc and anti-FLAG antibody separately. The endogenous K48- and K63-Ub were detected by Ub linkage specific antibodies. Immunoblots (IB) in ( a - e ) shows the indicated proteins detected with specific antibodies. WCL, whole cell lysate.

Article Snippet: Caspase-4 (Cat# 4450S, 1:1000), Gasdermin D (Cat# 39754S, 1:1000), Cleaved Gasdermin D (Asp275) (Cat# 36425S, 1:500), Cleaved Gasdermin D (Asp276) (Cat# 10137S, 1:1000), NLRP3 (Cat# 15101S, 1:1000), Tubulin (Cat# 2144S, 1:2000), Actin (Cat#4967S, 1:2000), UBXN3B (Cat# 34945S, 1:1000), K63-linkage specific polyubiquitin (Clone D7A11, Cat# 5621S,1:500), K48-linkage specific polyubiquitin (Clone D9D5, Cat# 12805S, 1:1000), Phospho-Jak1 (Tyr1034/1035) (Cat# 74129S, 1:1000), Phospho-Stat1 (Tyr701) (Cat# 9167S, 1:1000), Myc-Tag (Cat# 2276S, 1:2000) and HA-Tag (Clone C29F4, Cat# 3724S, 1:1000) antibodies were from Cell Signaling.

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Knock-Out, Mutagenesis

Journal: EMBO reports

Article Title: UBXD8 mediates mitochondria-associated degradation to restrain apoptosis and mitophagy.

doi: 10.15252/embr.202254859

Figure Lengend Snippet: Figure 4. UBXD8 degrades mitochondrial and endoplasmic reticulum (ER) substrates in cis and in trans.

Article Snippet: We obtained a monoclonal UBXD8 antibody (#34945, CST) for immunofluorescence staining.

Techniques:

Journal: EMBO reports

Article Title: UBXD8 mediates mitochondria-associated degradation to restrain apoptosis and mitophagy.

doi: 10.15252/embr.202254859

Figure Lengend Snippet: Figure 4. UBXD8 degrades mitochondrial and endoplasmic reticulum (ER) substrates in cis and in trans.

Article Snippet: Actin (Sigma-Aldrich, A2066), AIF (Santa Cruz Biotechnology, SC13116), Bad (Santa Cruz Biotechnology, SC-8044), Bak (Cell Signaling Technology, 3814), Bax (Cell Signaling Technology, 5023), Beclin1 (Cell Signaling Technology, 3495), Bcl-2 (Proteintech, 12789-1-AP), Bcl-xL (Cell Signaling Technology, 2764), Bid (Cell Signaling Technology, 2002), Bik (Santa Cruz Biotechnology, SC365625), Bim (Cell Signaling Technology, 2933), Blk (Santa Cruz Biotechnology, SC-65980), Bnip3 (Abcam, ab109362), Caspase-3 (Cell Signaling Technology, 9662), FLAG (Sigma-Aldrich, F1804) Hsp60 (Cell Signaling Technology, 4870), LonP1 (Proteintech, 15440-1-AP), MARCH5 (Cell Signaling Technology, 19168), Mcl1 (Santa Cruz Biotechnology, SC-819), Mfn1 (Proteintech, 13798-1- AP), Mfn2 (Cell Signaling Technology, 9482), MiD49 (Proteintech, 16413-1-AP), Nix (Cell Signaling Technology, 12396), Noxa (Santa Cruz Biotechnology, SC-56169), P4D1 (Santa Cruz Biotechnology, SC-8017), PARP (Cell Signaling Technology, 9542), Prohibitin (Proteintech, 12295-1-AP), Puma (Santa Cruz Biotechnology, SC374223), RNF185 (Abcam, ab181999), Smac (Cell Signaling Technology, 2954), Tim23 (Proteintech, 11123-1-AP), Tom22 (Proteintech, 11278-1-AP), Tom40 (Proteintech, 18409-1-AP), Tom70 (Proteintech, 14528-1-AP), UBXD2 (Proteintech, 21052-1-AP), UBXD8 (Cell Signaling Technology, 34945; Santa Cruz Biotechnology, SC-374098), VCP (Proteintech, 10736-1-AP), Donkey anti-rabbit IgG (Jackson ImmunoResearch, 711-035-152), Goat anti-mouse IgG (Sigma-Aldrich, A5278), HA-peroxidase (Sigma-Aldrich, H6533).

Techniques: