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