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
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