Journal: Biochimica et biophysica acta. Gene regulatory mechanisms

Article Title: The SAGA HAT module is tethered by its SWIRM domain and modulates activity of the SAGA DUB module.

doi: 10.1016/j.bbagrm.2023.194929

Figure Lengend Snippet: Fig. 4. SAGA's HAT module regulates SAGA DUB activity independent of enzymatic activity. A: Quantitation of western blots of SAGA DUB activity on recombinant H2B-Ub nucleosomes (400 nM) under conditions of limiting SAGA enzyme (25 nM) with and without the SWIRM domain. B: Quantitation of western blots of SAGA DUB activity on HeLa nucleosomes with ~50 nM H2B ubiquitinated nucleosome. SAGA DUB activity is compared with and without the SWIRM domain, with excess SAGA, 100 nM. C: WT SAGA DUB (25 nM) activity on recombinant H2B-Ub nucleosomes (400 nM) in absence and presence of acetyl CoA (10 μM). D: Quantitation of C and replicate.

Article Snippet: Experiments done with excess ubiquitinated nucleosome used purified recombinant mononucleosomes containing histone H2B ubiquitinated at K120 (H2B-K120Ub) (EpiCypher, 16-0370) at 400 nM and 25 nM SAGA.

Techniques: Activity Assay, Quantitation Assay, Western Blot, Recombinant

Journal: BMC Molecular Biology

Article Title: RNF168, a new RING finger, MIU-containing protein that modifies chromatin by ubiquitination of histones H2A and H2AX

doi: 10.1186/1471-2199-10-55

Figure Lengend Snippet: RNF168 resides in detergent-insoluble structures and interacts with the histone core . A) Before fixing, HeLa cells transfected with the GFP-tagged indicated constructs or the vector alone, were pre-treated (right panels) or not (left panels) with Triton X-100. The nucleus was stained with To-Pro 3. B) To test RNF168 capability to associate with histones, we performed in vitro pull down assay on cell lysate derived from 293T cells expressing the GFP-tagged forms of histones H2A, H2B, H3 and H4. To detect histone binding, the associated proteins were resolved by SDS-PAGE and analysed by anti-GFP immunoblot. To normalize for equal loading, Ponceau-red staining is shown.

Article Snippet: Antibodies used were: mouse monoclonal anti-FLAG and anti-FLAG affinity gel (M2, Sigma), mouse monoclonal anti RNF168 (Abcam), rabbit Phospho-(Ser/Thr) ATM/ATR Substrate Antibody (Cell Signaling), mouse monoclonal anti-Ub P4D1 (Santa Cruz) and FK2 (Stressgen Bioreagents), mouse monoclonal anti-ubiquityl-Histone H2A (Upstate), mouse monoclonal anti-ubiquityl-Histone H2B (Upstate), mouse monoclonal anti-GFP (Santa Cruz), rabbit polyclonal anti-GST was home made, anti phospho-Histone H2A.X (Ser139; Upstate).

Techniques: Transfection, Construct, Plasmid Preparation, Staining, In Vitro, Pull Down Assay, Derivative Assay, Expressing, Binding Assay, SDS Page, Western Blot

Journal: BMC Molecular Biology

Article Title: RNF168, a new RING finger, MIU-containing protein that modifies chromatin by ubiquitination of histones H2A and H2AX

doi: 10.1186/1471-2199-10-55

Figure Lengend Snippet: RNF168 ubiquitinates histone H2A but not H2B, both in vitro and in vivo . A) The in vitro ubiquitination assay was performed with GST-RNF168 wild type and the RF* mutant, using recombinant histones H2A and H2B as substrates. Refer to Methods for details. The reaction mixtures were solved by SDS-PAGE and the immunoblot with antibodies uH2A and uH2B shows the monoubiquitinated forms of histones H2A and H2B, respectively. TCL (Total Cell Lysates) are loaded to validate the signals. Immunoblots directed to RNF168 antibodies were done as control of equal loading. B) 293T cells were co-transfected with GFP alone, GFP-RNF168 WT or RF* together with FLAG-tagged histones H2A (left panels) or H2B (right panels). A small amount (1/20) of the transfected cells were lysed with standard procedure to verify protein expression level (IB using GFP and FLAG antibodies, lower panels). The remaining part was subjected to acid extraction, and the histone component was analysed by SDS-PAGE and immunodecorated as indicated. (◆) indicates the mono-ubiquitinated form of histones H2A and H2B; (◆) di- and (◆◆◆) tri-ubiquitinated forms are visible when the wild type RNF168 is expressed, but not in the presence of the RF* mutant or the vector alone. No signal of di- and tri-ubiquitination was detected for H2B. C) Cells transfected with the indicated GFP constructs were immunostained with anti-uH2A. D) 293T cells were co-transfected with cDNA encoding the GFP-RNF168 (WT) or the vector alone and the FLAG-H2A, and subjected to acid extraction. Proteins were resolved by SDS-PAGE and immunoblotted with FK2 (left panel) and Apu3.A8 (K63-chain specific, right panel) anti-Ub antibodies.

Article Snippet: Antibodies used were: mouse monoclonal anti-FLAG and anti-FLAG affinity gel (M2, Sigma), mouse monoclonal anti RNF168 (Abcam), rabbit Phospho-(Ser/Thr) ATM/ATR Substrate Antibody (Cell Signaling), mouse monoclonal anti-Ub P4D1 (Santa Cruz) and FK2 (Stressgen Bioreagents), mouse monoclonal anti-ubiquityl-Histone H2A (Upstate), mouse monoclonal anti-ubiquityl-Histone H2B (Upstate), mouse monoclonal anti-GFP (Santa Cruz), rabbit polyclonal anti-GST was home made, anti phospho-Histone H2A.X (Ser139; Upstate).

Techniques: In Vitro, In Vivo, Ubiquitin Proteomics, Mutagenesis, Recombinant, SDS Page, Western Blot, Control, Transfection, Expressing, Extraction, Plasmid Preparation, Construct

Journal: eLife

Article Title: Matrin 3-dependent neurotoxicity is modified by nucleic acid binding and nucleocytoplasmic localization

doi: 10.7554/eLife.35977

Figure Lengend Snippet:

Article Snippet: After electrophoresis, samples were transferred at 30 V overnight at 4°C onto an activated 2 μm nitrocellulose membrane (Bio-Rad), blocked with 3% BSA in 0.2% Tween-20 in Tris-buffered saline (TBST), and blotted overnight at 4°C with the following primary antibodies: rabbit anti-MATR3 (Abcam EPR10634(B)), mouse anti-GAPDH (Millipore Sigma MAB374; RRID: AB_2107445 ), and rabbit anti-H2B (Novus Biologicals NB100-56347; RRID: AB_838347 ), all diluted 1:1000 in 3% BSA, 0.2% TBST.

Techniques: Recombinant