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: World Journal of Surgical Oncology

Article Title: Interfering with UBE2L3 expression targets regulation of MLKL to promote necroptosis inhibition of growth in osteosarcoma

doi: 10.1186/s12957-025-03715-3

Figure Lengend Snippet: UBE2L3 knockdown inhibits tumor growth in vivo. ( A ) Representative images of tumors excised from mice injected with control (sh-NC) or UBE2L3 knockdown (sh-UBE2L3#1, sh-UBE2L3#2) cells. ( B ) Tumor volume was measured over 15 days. Tumors in the UBE2L3 knockdown groups grew significantly slower compared to the control group. ( C ) Tumor weight at the endpoint. Tumors in the UBE2L3 knockdown groups were significantly lighter than those in the control group. Data are shown as mean ± SD. *** P < 0.001

Article Snippet: For co-immunoprecipitation (Co-IP), equal amounts of protein were incubated overnight at 4 °C with UBE2L3 antibody (Proteintech, 13641-1-AP) or MLKL antibody (Abcam, ab184718) pre-bound to Protein A/G beads (Santa Cruz, sc-2003).

Techniques: Knockdown, In Vivo, Injection, Control

Journal: World Journal of Surgical Oncology

Article Title: Interfering with UBE2L3 expression targets regulation of MLKL to promote necroptosis inhibition of growth in osteosarcoma

doi: 10.1186/s12957-025-03715-3

Figure Lengend Snippet: UBE2L3 knockdown affects cell proliferation and necroptosis markers in tumor tissues. ( A ) Representative IHC staining of PCNA and quantification of PCNA-positive areas in NC and UBE2L3 knockdown groups. ( B ) Representative IHC staining of p-MLKL and quantification of p-MLKL-positive areas. ( C ) Representative IHC staining of p-RIPK1 and quantification of p-RIPK1-positive areas. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For co-immunoprecipitation (Co-IP), equal amounts of protein were incubated overnight at 4 °C with UBE2L3 antibody (Proteintech, 13641-1-AP) or MLKL antibody (Abcam, ab184718) pre-bound to Protein A/G beads (Santa Cruz, sc-2003).

Techniques: Knockdown, Immunohistochemistry

Journal: World Journal of Surgical Oncology

Article Title: Interfering with UBE2L3 expression targets regulation of MLKL to promote necroptosis inhibition of growth in osteosarcoma

doi: 10.1186/s12957-025-03715-3

Figure Lengend Snippet: UBE2L3 knockdown activates the necroptosis signaling pathway. ( A ) Western blot analysis of p-RIPK1, RIPK1, p-MLKL, and MLKL protein levels in NC and UBE2L3 knockdown groups. GAPDH served as the loading control. ( B ) Quantification of protein expression levels normalized to GAPDH, including the ratios of p-RIPK1/RIPK1 and p-MLKL/MLKL. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01

Article Snippet: For co-immunoprecipitation (Co-IP), equal amounts of protein were incubated overnight at 4 °C with UBE2L3 antibody (Proteintech, 13641-1-AP) or MLKL antibody (Abcam, ab184718) pre-bound to Protein A/G beads (Santa Cruz, sc-2003).

Techniques: Knockdown, Western Blot, Control, Expressing

Journal: World Journal of Surgical Oncology

Article Title: Interfering with UBE2L3 expression targets regulation of MLKL to promote necroptosis inhibition of growth in osteosarcoma

doi: 10.1186/s12957-025-03715-3

Figure Lengend Snippet: Western blot validation of the binding between UBE2L3 and MLKL

Article Snippet: For co-immunoprecipitation (Co-IP), equal amounts of protein were incubated overnight at 4 °C with UBE2L3 antibody (Proteintech, 13641-1-AP) or MLKL antibody (Abcam, ab184718) pre-bound to Protein A/G beads (Santa Cruz, sc-2003).

Techniques: Western Blot, Biomarker Discovery, Binding Assay

Journal: World Journal of Surgical Oncology

Article Title: Interfering with UBE2L3 expression targets regulation of MLKL to promote necroptosis inhibition of growth in osteosarcoma

doi: 10.1186/s12957-025-03715-3

Figure Lengend Snippet: UBE2L3 promotes ubiquitination modification of MLKL

Article Snippet: For co-immunoprecipitation (Co-IP), equal amounts of protein were incubated overnight at 4 °C with UBE2L3 antibody (Proteintech, 13641-1-AP) or MLKL antibody (Abcam, ab184718) pre-bound to Protein A/G beads (Santa Cruz, sc-2003).

Techniques: Ubiquitin Proteomics, Modification

Journal: World Journal of Surgical Oncology

Article Title: Interfering with UBE2L3 expression targets regulation of MLKL to promote necroptosis inhibition of growth in osteosarcoma

doi: 10.1186/s12957-025-03715-3

Figure Lengend Snippet: Immunoprecipitation confirms the interaction between UBE2L3 and MLKL

Article Snippet: For co-immunoprecipitation (Co-IP), equal amounts of protein were incubated overnight at 4 °C with UBE2L3 antibody (Proteintech, 13641-1-AP) or MLKL antibody (Abcam, ab184718) pre-bound to Protein A/G beads (Santa Cruz, sc-2003).

Techniques: Immunoprecipitation