constructs for uba1  (Addgene inc)

Journal: bioRxiv

Article Title: TRIM32–UBQLN2–p62 axis promotes TDP-43 inclusion formation and amyloid aggregation through shuttle condensates

doi: 10.64898/2026.02.11.705390

Figure Lengend Snippet: (A) Schematic of human TRIM32 highlighting domain architecture. The four lysine residues in the coiled-coil domain identified by mass spectrum analysis as autoubiquitination sites in the current study are indicated in red. The hydrophobic helix loop in TRIM32 NHL domain is labeled in blue. (B) Depiction of the core components of the TRIM32 in vitro auto-ubiquitination reconstitution assay including UBE1, the E2 enzyme UBE2D3, ubiquitin, TRIM32, and ATP. (C) Coomassie stained SDS-PAGE gels of in vitro TRIM32 autoubiquitination assays showing that compared with WT TRIM32, the quadruple point mutation TRIM32 4KR (K175R/K182R/K204R/K215R) prevents autoubiquitination. Colored box indicates the band for TRIM32 (cyan) and ubiquitin (blue) level quantification, respectively. Red arrow indicates HWM polyubiquitinated TRIM32 in the loading well. (D) Quantification of time course change in (C) measuring TRIM32 or ubiquitin level via densitometry. Combined data from three independent experiments are shown. Data are presented as mean ± s.e.m. (E) Ubiquitin linkage composition by AQUA MS from in vitro TRIM32 autoubiquitination assays performed in technical duplicate. (F) Coomassie stained SDS-PAGE gel image representing TRIM32 autoubiquitination assays before and after Lb pro * treatment for 1 hours. Released ubiquitin (blue box) was analyzed by MS to identify GG-modified ubiquitin species. (G) TRIM32 assembles branched polyUb, according to intact MS analysis. Quantification by spectra deconvolution of individual ubiquitin species from (F) in autoubiquitination assay.

Article Snippet: Bacterial plasmids obtained from Addgene include: UBE1 (Addgene plasmid # 34965), UBE2D3 (Addgene plasmid # 12643), ANXA11 (Addgene plasmid # 164496), p62 (Addgene plasmid # 190929), TDP-43 (Addgene plasmid # 133320 and plasmid # 27462).

Techniques: Labeling, In Vitro, Ubiquitin Proteomics, Reconstitution Assay, Staining, SDS Page, Mutagenesis, Modification

Journal: The EMBO Journal

Article Title: Ubiquitin is directly linked via an ester to protein-conjugated mono-ADP-ribose

doi: 10.1038/s44318-025-00391-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: pET21d-UBE1 ( H. sapiens ) , , Addgene #34965A.

Techniques: Control, Recombinant, Binding Assay, Ubiquitin Proteomics, Transfection, Protease Inhibitor, Synthesized, Silver Staining, Software, Imaging