Journal: Research

Article Title: USP18 Antagonizes Pyroptosis by Facilitating Selective Autophagic Degradation of Gasdermin D

doi: 10.34133/research.0380

Figure Lengend Snippet: E3 ubiquitin ligase MIB2 enhances GSDMD degradation via mediating the ubiquitination of GSDMD at K168. (A and B) HEK293T cells were transfected with HA-GSDMD and increasing amounts of Flag-MIB2. Cell lysates were collected for immunoblot analysis (A). Quantification of the relative protein level of GSDMD was shown in (B). (C to F) THP-1-derived macrophages were transfected with control siRNA (si Ctrl ) or MIB2 siRNA (si MIB2 ) for 48 h, pre-treated with LPS (200 ng/ml) for 3 h, then treated with DMSO (vehicle), MG132 (10 μM) or carfilzomib (100 nM), 3-methyladenine (3-MA, 10 mM), bafilomycin A1 (Baf A1, 0.2 μM), or chloroquine (CQ, 50 μM) for 6 h. Cell lysates were collected for immunoblot analysis (C and E). Quantitative analysis of the relative protein level of GSDMD was shown in (D) and (F), respectively. (G and H) THP-1-derived macrophages were transfected with si Ctrl or si MIB2 (#1 and #2) for 48 h, then primed with LPS (200 ng/ml) for 3 h and followed by ATP (5 mM, 3 h) treatment. Cell lysates were collected for immunoprecipitation and immunoblot analysis (G). Quantitative analysis of the relative immunoprecipitated level of ubiquitin (Ub) was shown in (H). (I and J) HEK293T cells were transfected with HA-Ub and wild type (WT) Flag-GSDMD or Flag-GSDMD K168R, together with Myc-empty vector (EV) or Myc-MIB2 for 18 h, then treated with Baf A1 (0.2 μM) for 6 h. Cell lysates were collected for immunoprecipitation and immunoblot analysis (I). Quantitative analysis of the relative immunoprecipitated level of HA-Ub was shown in (J). (K and L) Immunoblot analysis of HEK293T cells transfected with Flag-GSDMD WT or Flag-GSDMD K168R mutant, together with HA-EV or HA-MIB2 (K). Quantitative analysis of the relative protein level of GSDMD was shown in (L). (M) In vitro binding assay of 6×His-MIB2 and Flag-GSDMD proteins purified from HEK293T cells, respectively. (N) In vitro ubiquitination assay was performed in the presence of Ub, E1, E2 (UbcH5a), Flag-GSDMD, and 6×His-MIB2. (O to Q) THP-1-derived macrophages were transfected with si Ctrl or si MIB2 (#1 and #2) for 48 h, then primed with LPS (200 ng/ml) for 3 h and followed by ATP (5 mM, 3 h) treatment. Cell lysates were collected for immunoblot analysis (O). Cell death (P) and production of IL-1β (Q) were assessed by LDH (lactate dehydrogenase) release assay and ELISA analysis in the supernatants, respectively. In (A), (C), (E), (G), (I), (K) and (M) to (O), data are representative of 3 independent experiments with similar results. In (B), (D), (F), (H), (J), and (L), quantification of the indicated protein levels was determined by Image Lab software, data are presented as mean values ± SD, and P values were determined by unpaired 2-tailed Student’s t test of n = 3 independent biological experiments. In (P) and (Q), data are presented as mean values ± SEM and P values were determined by unpaired 2-tailed Student’s t test of n = 3 independent biological experiments.

Article Snippet: Briefly, purified Flag-GSDMD was incubated with ubiquitin (Ub), E1 activating enzyme, Mg-ATP, and E2 enzyme (UbcH5a, MCE, Cat# HY-P71399) with or without purified 6×His-MIB2 protein in ubiquitinoylation buffer in a total 50-μl reaction volume at 37 °C for 3 h. The assays were quenched by the addition of 50 μl of 2× non-reducing gel loading buffer, and 20 μl of each quenched assay was resolved by SDS-PAGE.

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Derivative Assay, Control, Immunoprecipitation, Plasmid Preparation, Mutagenesis, In Vitro, Binding Assay, Purification, Release Assay, Enzyme-linked Immunosorbent Assay, Software

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