Journal: Nature communications

Article Title: UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168.

doi: 10.1038/s41467-024-49431-6

Figure Lengend Snippet: Fig. 6 | UBE2D3 promotes KAP1 phosphorylation and telomere NHEJ in a PP2A- dependent manner. a Immunoblotting for pKAP1 (S824) in TRF2ts MEFs trans- duced with control, Ube2d3 and/or two independent Ppp2ca shRNAs at 32 °C or after 3 h at 37 °C. Representative blots from 2 independent experiments. b Quantification of chromosome fusions in TRF2ts MEFs transduced with control, Ube2d3 and two independent Ppp2ca shRNAs, upon 24 h of telomere uncapping. Two independent experiments are shown. c PP2A activity assays with immuno- precipitated PP2A from TRF2ts MEFs transduced as indicated and cultured at 32 °C or for 3 h at 37 °C to induce telomere uncapping (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). Immunoblots of input and immunopre- cipitates are shown in Supplementary Fig. 8e. d PP2A phosphatase activity assay with immunoprecipitated PP2A-alpha (PP2Ac) from RNF168 mutant human cells (RIDDLE) with and without expression of ectopic HA-RNF168. Corrected for the amount of immunoprecipitated PP2A-alpha. Cells were untreated or harvested 30 min after irradiation with 3 Gy (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). The different symbols (dot, square and triangle)

Article Snippet: Primary antibodies used were against UBE2D3 (Y-25, sc-100618, SCBT, 1:500; 11677-1-AP, Proteintech, 1:500; 4330S, CST, 1:500 and A615, Boston Biochem, 1:2000), KAP1 (22553, Abcam, 1:1000), phospho-Kap1 S824 (A300 767A, Bethyl, 1:1000), 53BP1 (NB100-305, Novus, 1:500 and A300-272A, Bethyl, 1:2000), phospho-ATMS1981 (4526, CST, 1:1000), phospho-H2AX S139 (5636, Millipore, 1:1000), CHK2 (611570, BD, 1:500), c-myc (9E10, sc-40, SCBT, 1:250), HA (MMS101R, Covance, 1:1000), TRF2 (NB110-57130, Novus, 1:500), RNF8 (sc133971, SCBT, 1:250), MAD2L2 (14, sc-135977, SCBT, 1:500), GFP IgG fraction (A11122, Thermo Fisher Scientific, 1:1000), FLAG M2 (F1804, Sigma-Aldrich, 1:1000), Histone H3 (ab1791, Abcam, 1:10,000), hRNF168 (ABE367, Millipore, 1:500), hRNF168 (ABE467, Merck-Millipore, 1:1000), mRnf168 (gift from D. Durocher, 1:1000), hRIF1 (A300569A, Bethyl, 1:1000), mRIF1 (gift from S. Boulton and R. Chapman, 1:1000), Ligase 4 (H-300, sc-28232, SCBT, 1:300; NB110-57379, Novus, 1:500), FK2 (04-263, Millipore, 1:2000), HP1α (2616S, CST, 1:1000), Ubiquitin (P4D1, sc-8017, SCBT, 1:1000), HDAC1 (PA1-860, Thermo Fisher Scientific, 1:1000), PP2A C subunit, clone 1D6 antibody (05-421, Sigma-Aldrich/Millipore, 1:500), CDK4 (C-22, sc-260, SCBT, 1:500), HSP90 α/β (H-114, sc-7947, SCBT, 1:1000), γ-tubulin (T6557, SigmaAldrich, 1:10,000) β-actin (A5316, Sigma-Aldrich, 1:10,000), β-catenin (610154, BD, 1:10,000) and GAPDH (PA1-987, Thermo Fisher Scientific, 1:1000).HSP90α/β, γ-tubulin,β-actin,β-catenin andGAPDHwere used for loading controls.

Techniques: Phospho-proteomics, Western Blot, Control, Transduction, Activity Assay, Cell Culture, Two Tailed Test, Phosphatase Assay, Immunoprecipitation, Mutagenesis, Expressing, Irradiation

Journal: International journal of molecular medicine

Article Title: Identification of NR0B1 as a novel androgen receptor co-repressor in mouse Sertoli cells.

doi: 10.3892/ijmm.2016.2672

Figure Lengend Snippet: Figure 4. Inhibition of androgen receptor (AR) target genes by NR0B1 in an androgen-dependent manner. TM4 cells were transfected with AR- and NR0B1‑overexpressing plasmids. Following treatment with 10 nM testosterone (T) for 24 h, the expression of ubiquitin-conjugating enzyme E2B (UBE2B) and heat shock transcription factor 1 (HSF1) in the TM4 cells was examined by (A and B) RT-qPCR and (C and D) western blot analysis. Data are expressed as the means ± SD (n=5), and bars marked with asterisks indicate significant differences (P<0.05). All experiments were performed three times by independent individuals. AR, only AR-overexpressing plasmid; AR+T, AR-overexpressing plasmid and T; AR+T+NR0B1, AR-overexpressing plasmid and T and NR0B1-overexpressing plasmid; AR+T+NR0B1+SiNr0b1, AR-overexpressing plasmid and T and NR0B1-overexpressing plasmid and siRNA-mediated knockdown NR0B1.

Article Snippet: Following incubation with the anti-NR0B1 antibody (1:1,000; #ab60144; Abcam), anti-UBE2B antibody (1:2,000; #10733-1-AP; Proteintech, Chicago, IL, USA) and anti-HSF1 antibody (1:2,000; #ABE1044; Millipore Corp.) overnight at 4 ̊C, the membranes were treated with HRP-labeled secondary antibody (ab6721; Abcam) for 1 h at room temperature.

Techniques: Inhibition, Transfection, Expressing, Ubiquitin Proteomics, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Knockdown

Journal: Frontiers in Cardiovascular Medicine

Article Title: Nutrient restriction protects against valve interstitial cell calcification by upregulating ubiquitin mediated proteolysis

doi: 10.3389/fcvm.2025.1586775

Figure Lengend Snippet: Proteomic analysis shows the upregulated UPS proteins in nutrient-deprived RVICs. (A) Fold change and function of proteins involved in ubiquitin proteasome pathway. (B) IPA network analysis shows altered activation of key UPS pathway proteins including Cul2, Ube2H, SKP1 and Ubiquitin highlighted in blue circle. Functional interconnections between the proteins are shown by arrows and lines. The blue lines represent direct associations with Annexin VI. Dashed lines represent predicted associations. (C–E) Validation and quantification of Cul2 and Ube2h expression by immunoblots. Equal amounts of total protein were loaded in each lane, and all blots were processed in parallel under identical experimental conditions to ensure comparability. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary antibodies included those against Runx2 (Abcam, ab236639), Cul2 (Proteintech, 10981-2-AP), Ube2h (Proteintech, 15685-1-AP), OCN (Proteintech, 23418-1-AP), Osterix (Proteintech, 28694-1-AP), β-actin (Proteintech, 66009-1-Ig) and bone sialoprotein (Invitrogen, PA5-79425).

Techniques: Ubiquitin Proteomics, Activation Assay, Functional Assay, Biomarker Discovery, Expressing, Western Blot