e2 e2 640 r d systems  (R&D Systems)

Journal: Leukemia

Article Title: Distinct characteristics of VEXAS-causative UBA1 M41 and recurrent functional non-M41 mutations

doi: 10.1038/s41375-025-02775-4

Figure Lengend Snippet: A Schematic representation of the UBA1 gene, highlighting its functional domains and pinpointing the exact locations of the identified UBA1 variants. The variants were classified into Tier-1 (pathogenic: P), Tier-2 (likely pathogenic: LP) and Tier-3 (variant of uncertain significance: VUS), as depicted in the outer pie chart on the left. UBA1 variants are displayed on top of the gene if the locus occurred recurrently in our screen. Combination of variants detected in the same patients are also shown. Variants detected only as combinations are italicized. M41 and other tier assignments are color-coded. Bold underlined variants were tested for functional significance. B Chart summarizing UBA1 variants and their functional status, indicating the presence or absence of ubiquitylation. Quantification of polyubiquitin levels and mono-ubiquitylated histone H2A/B (Ub-H2A, Ub-H2B) or E2 enzymes (UBE2D3-Ub, UBE2L3-Ub) were normalized within each sample to β-actin levels and scaled to WT transfection. E2 enzyme ubiquitylation was quantified as the ratio of charged form to uncharged form and scaled to WT. Data represent n = 3–6 biological replicates, shown as mean −/+ s.d., significance determined by unpaired t-test with Welch’s correction (*p < 0.05, **p < 0.01).

Article Snippet: Primary antibodies for Poly-ubiquitin (Cell Signaling, 3936S), UBE2D3 (Cell-Signaling, 4330), UBE2L3 (R&D Systems, E2-640), ubiquitylated-H2A (Cell Signaling, 8240), H2A (Cell Signaling, 12349), ubiquitylated-H2B (Cell Signaling, 5546S), H2B (Cell Signaling, 12364), and β-actin (Cell Signaling, 4970) were used at a concentration of 1:1000 and visualized using HRP-conjugated secondary antibodies (anti-rabbit [Cell Signaling, 7074S] or anti-mouse [Cell Signaling, 7076S]) at a concentration of 1:3000.

Techniques: Functional Assay, Variant Assay, Transfection

Journal: iScience

Article Title: Trichinella spiralis inhibits myoblast differentiation by targeting SQSTM1/p62 with a secreted E3 ubiquitin ligase

doi: 10.1016/j.isci.2024.109102

Figure Lengend Snippet:

Article Snippet: Ubiquitin E2 Enzyme UbcH7 , Boston Biochem, USA , Cat#E2-640.

Techniques: Recombinant, Ubiquitin Proteomics, Conjugation Assay, Transfection, Plasmid Preparation, Software

Journal: The Journal of biological chemistry

Article Title: β-arrestin1 is an E3 ubiquitin ligase adaptor for substrate linear polyubiquitination.

doi: 10.1016/j.jbc.2023.105474

Figure Lengend Snippet: Figure 1. In vitro reconstitution of STAM1 ubiquitination by AIP4. A, STAM1 ubiquitination by AIP4 was reconstituted in vitro and performed in the presence of increasing concentrations of βarrestin1. Ubiquitination reactions comprised of E1(Ube1, 42 nM), E2 (UbcH7, 350 nM), E3 (AIP4, 48.5 nM), ubiquitin (11.6 μM), DTT (1 mM), ATP (1 mM) plus STAM1 (42 nM), and varying concentrations of βarr1 (0 nM, 20 nM, 40 nM, 80 nM, 160 nM, or 320 nM) in 40 μl. Reactions were incubated for 90 min at 37 C and terminated with 40 μl 2× sample buffer. Equal volumes were analyzed by 7% SDS-PAGE and immunoblotting with the indicated antibodies. Polyubiquitinated [Ub(n)] and unmodified STAM1 are indicated. Immunoblots are from one representative experiment. STAM1 ubiquitination was quantified from the STAM (B) and ubiquitin (C) immunoblots using densitometry and shown as line graphs. Data were expressed relative to the signal at 300 nM βarr1 and represent the mean ± S.D. from three independent experiments. Curves were fitted by nonlinear regression, Michaelis–Menten (GraphPad Prism). AIP4, atrophin-interacting protein 4; STAM, signal-transducing adaptor molecule.

Article Snippet: UbcH7 (E2; cat# E2-640–100), lysine-less ubiquitin (0K-Ub; cat# UM-NOK) and lysine-less biotinylated ubiquitin (0K-Ub biotin; cat# UB-560) were from R&D Systems.

Techniques: In Vitro, Ubiquitin Proteomics, Incubation, SDS Page, Western Blot