Journal: Communications Biology

Article Title: Fanconi anemia associated protein 20 (FAAP20) plays an essential role in homology-directed repair of DNA double-strand breaks

doi: 10.1038/s42003-023-05252-9

Figure Lengend Snippet: a Quantification of 2 Gy IR-treated cells with indicated knockdown conditions containing 5 or more 53BP1 foci reported as a % of total cells analyzed. b Identical to 5a but showing quantification of BRCA1 IRIF foci. c Identical to Fig. but showing quantification of RAD51 IRIF foci. d Representative cell images from immunofluorescent (IF) staining in U2OS cells. Cells were transfected with the indicated siRNAs and then treated with 2 Gy IR 6 h before harvesting and staining. RAD51 is recognized by anti-rabbit Alexa-488; γH2AX antibody is recognized by anti-mouse Alexa-594; and cell boundaries were determined with DAPI signal. Scale bars are shown at 10 μM. e Identical to Fig. but showing quantification of γH2AX IRIF. f Cell cycle distribution analysis by propidium iodide (PI) staining in U2OS cells with the same treatment conditions as IF staining conditions shown in Fig. . For graphs ( a – c , e , f ), bars show mean with error bars as SD. Two-tailed Student’s t test were used for statistical analysis where ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, n = 3 for all experiments. g Chromatin fractionation and western blotting of chromatin fraction vs whole cell extract for RAD51 with the indicated protein knockdown. Cell conditions matched those used in IF staining experiments (2 Gy IR-treated U2OS cells, harvested after 6 h). 40 μg protein was loaded for each sample. H2B protein was used as a loading control for the chromatin-bound fraction, while HSP90 was used as a loading control for whole cell extract. One representative gel from three individual experiments is shown. h Coimmunoprecipitation in HEK293t whole cell extracts that overexpress WT FAAP20, using anti-RAD51 IgG and with Rb IgG isotype control. 53BP1 was probed as a negative control. n = 3.

Article Snippet: Gels were run 1–1.5 h at 150 V then transferred to nitrocellulose membranes, blocked for 1 h with 5% milk, then probed with the following antibodies: FANCA (Bethyl-1:1000), FAAP20 (Sigma, Weidong Wang-1:250), FANCG (Santa Cruz-1:50), Actin (Santa Cruz-1:2000), H2B (Cell Signaling Technologies-1:1000), BRCA2 (Sigma, OHSU-1:500), RAD51 (BioAcademia-1:500), HSP90 (Santa Cruz-1:2000), FANCD2 (Proteintech-1:1000).

Techniques: Staining, Transfection, Two Tailed Test, Fractionation, Western Blot, Negative Control

Journal: iScience

Article Title: Inositol polyphosphate multikinase modulates redox signaling through nuclear factor erythroid 2-related factor 2 and glutathione metabolism

doi: 10.1016/j.isci.2023.107199

Figure Lengend Snippet: IPMK binds Nrf2 and inhibits its activity (A) IPMK interacts with Nrf2. HEK293 cells were transfected with GFP-Nrf2 and either empty GST vector or GST-IPMK for 24 h, following which cells were harvested and GST-pulldown conducted using GSH Sepharose beads and analyzed by western blotting using anti-GFP Nrf2. (B) Nuclear Nrf2 is decreased by IPMK. A representative western blot showing the distribution of GFP-Nrf2 in the nuclear and cytoplasmic compartment. HEK293 cells were transfected with GFP-Nrf2 and either empty GST vector or GST-IPMK for 24 h, following which cells were harvested and nuclear and cytoplasmic fractions analyzed for localization of Nrf2 by western blotting with anti-GFP antibodies. Anti-histone H2B (nuclear marker) and lactate dehydrogenase (LDH, cytoplasmic marker) were utilized to assess the purity of the samples. In the presence of IPMK, nuclear localization of GFP-Nrf2 is diminished. (C) Quantitation of relative levels of GFP-Nrf2 in the nuclear and cytoplasmic compartments. ∗∗p < 0.01, Two-tailed Student’s t test (n = 3, mean ± SEM). (D) Catalytic activity of IPMK is not required for diminishing expression of Nrf2 targets. Lysates from wild-type MEFs, IPMK null cells and IPMK null cells complemented either with wild-type IPMK or catalytically inactive KSA mutant were prepared and expression of the Nrf2 targets analyzed. IPMK inhibited the expression of the tested Nrf2 target genes in a catalytically independent manner. (E–H) The suppression of Nrf2 targets, Gclc , GstA2 , Hmox1 and Txnrd1 occur at the transcriptional level. ∗∗∗∗p < 0.0001, Two-tailed Student’s t test (n = 3, mean ± SEM). (I) Model depicting IPMK action. In wild-type cells, IPMK regulates Nrf2 activity by binding to it and inhibiting its nuclear translocation. As a result, when IPMK is depleted, increased nuclear translocation of Nrf2 occurs, leading to elevated expression of target genes.

Article Snippet: Anti-H2B (1:1000) , Cell Signaling , Cat# 8135, RRID:AB_10891053.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Western Blot, Marker, Quantitation Assay, Two Tailed Test, Expressing, Mutagenesis, Binding Assay, Translocation Assay

Journal: iScience

Article Title: Inositol polyphosphate multikinase modulates redox signaling through nuclear factor erythroid 2-related factor 2 and glutathione metabolism

doi: 10.1016/j.isci.2023.107199

Figure Lengend Snippet:

Article Snippet: Anti-H2B (1:1000) , Cell Signaling , Cat# 8135, RRID:AB_10891053.

Techniques: Recombinant, Modification, Transfection, Protease Inhibitor, Fractionation, Glutathione Assay, Expressing, Concentration Assay, Plasmid Preparation, Construct, Software

Journal: bioRxiv

Article Title: A system for in vivo evaluation of protein ubiquitination dynamics using deubiquitinase-deficient strains

doi: 10.1101/2023.06.18.545485

Figure Lengend Snippet: (A-B) Immunoblots for histone H2BK123ub1 and/or histone H3K4 methylation (mono, me1; di, me2; tri, me3) in extracts prepared from A) strains lacking Rad6, Bre1 or Lge1 and B) strains lacking Rad6, Bre1 or Lge1 in the background of ubp8Δubp10Δ double deletion mutant. Ponceau S staining and histone H3 levels served as loading controls. Histone H2BK123ub1 was detected using anti-H2B or anti-H2BK120 ubiquityl antibody. Molecular weights of the protein standards used as size markers (kDa) are indicated.

Article Snippet: The following antibodies were used in immunoblotting: anti-Flag M2 (F3165; Sigma), anti-V5 (46-0708; Invitrogen); anti-HA (39628; Active Motif); anti-Pgk1 (459250; Invitrogen), anti-H2B (39237; Active Motif), anti-H3 (ab1791; Abcam), anti-H3K4me1 (39297; Active Motif), anti-H3K4me2 (399141; Active Motif), anti-H3K4me3 (39159; Active Motif), anti-ubiquityl-Histone H2B (Lys120) (D11) XP® (5546; Cell Signaling); anti-mono- and polyubiquitinylated conjugates monoclonal antibody (clone FK2) (HRP conjugate) (BML-PW0150; Enzo Life Sciences), anti-PCNA/Pol30 (ab221196; Abcam).

Techniques: Western Blot, Methylation, Mutagenesis, Staining

Journal: bioRxiv

Article Title: A system for in vivo evaluation of protein ubiquitination dynamics using deubiquitinase-deficient strains

doi: 10.1101/2023.06.18.545485

Figure Lengend Snippet: (A) Sequences of the distal end of WT H2B C-terminal helix and of mutants. Lysine 123, the site of monoubiquitination is indicated. (B) Growth assay conducted by spotting 10-fold serial dilutions of indicated strains on synthetic medium lacking histidine (-HIS) or lacking histidine and containing 5-fluoroorotic acid (-HIS+FOA). (C-D) Left: Immunoblots for H2BK123ub1 in extracts prepared from C) UBP8UBP10 or D) ubp8Δubp10Δ strains expressing Flag epitope-tagged WT or mutant histone H2B. Ponceau S staining served as loading control. Right: Fold-change in H2BK123ub1 levels in the indicated mutants relative to WT H2B (set as 1). For densitometry quantitation, the signals for H2BK123ub1 in WT or mutant H2B were initially normalized to the signals for Ponceau S-stained proteins. Plotted are means ± SEM from three independent experiments. ns, not significant; *, p -value <0.05 (Student’s t-test).

Article Snippet: The following antibodies were used in immunoblotting: anti-Flag M2 (F3165; Sigma), anti-V5 (46-0708; Invitrogen); anti-HA (39628; Active Motif); anti-Pgk1 (459250; Invitrogen), anti-H2B (39237; Active Motif), anti-H3 (ab1791; Abcam), anti-H3K4me1 (39297; Active Motif), anti-H3K4me2 (399141; Active Motif), anti-H3K4me3 (39159; Active Motif), anti-ubiquityl-Histone H2B (Lys120) (D11) XP® (5546; Cell Signaling); anti-mono- and polyubiquitinylated conjugates monoclonal antibody (clone FK2) (HRP conjugate) (BML-PW0150; Enzo Life Sciences), anti-PCNA/Pol30 (ab221196; Abcam).

Techniques: Growth Assay, Western Blot, Expressing, FLAG-tag, Mutagenesis, Staining, Quantitation Assay

Journal: bioRxiv

Article Title: A system for in vivo evaluation of protein ubiquitination dynamics using deubiquitinase-deficient strains

doi: 10.1101/2023.06.18.545485

Figure Lengend Snippet: (A) The steady-state levels of ubiquitination of a protein in vivo are maintained by the actions of ‘writer’ E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases and ‘eraser’ DUBs. (B) In yeast S. cerevisiae , the in vivo steady-state levels of H2BK123ub1 in a nucleosome are maintained by the ‘writer’ complex comprised of Rad6 (E2), Bre1 (E3) and accessory/adapter protein Lge1, and two ‘eraser’ DUBs, Ubp10 and the SAGA complex-associated Ubp8. IDR, intrinsically disordered region; cc, coiled-coil domain. (C) In the ubp8Δubp10Δ double null mutant strain, H2BK123ub1 accumulates due to ubiquitin addition and absence of deubiquitination. (D) High levels of H2BK123ub1 are not observed in the ubp8Δubp10Δ mutant strain when either the IDR or coiled-coil domain of Lge1 or the C-terminal acidic tail of Rad6 are deleted. Thus, the absence of relevant DUBs revealed the roles for various regions or domains of proteins involved in the ubiquitin-conjugation step. (E) Residues of the H2B C-terminal helix (Cα) impact the activity of the E2-E3 complex and the DUBs by influencing their access to substrate K123 or its ubiquitin conjugated form, respectively. Aspartate substitution at residue 120 in H2B Cα inhibits the Rad6-Bre1-Lge1-mediated monoubiquitination of H2BK123 in both WT and ubp8Δubp10Δ strains. In contrast, arginine substitution at position 120 in H2B Cα promotes removal of the conjugated ubiquitin by Ubp8 and Ubp10, as evidenced by the reduced H2BK123ub1 in the H2BA120R mutation in a strain expressing these two DUBs and not in their absence. Thus, the use of the DUB deletion strain informed on the dynamics of deubiquitination in addition to the ubiquitin conjugation.

Article Snippet: The following antibodies were used in immunoblotting: anti-Flag M2 (F3165; Sigma), anti-V5 (46-0708; Invitrogen); anti-HA (39628; Active Motif); anti-Pgk1 (459250; Invitrogen), anti-H2B (39237; Active Motif), anti-H3 (ab1791; Abcam), anti-H3K4me1 (39297; Active Motif), anti-H3K4me2 (399141; Active Motif), anti-H3K4me3 (39159; Active Motif), anti-ubiquityl-Histone H2B (Lys120) (D11) XP® (5546; Cell Signaling); anti-mono- and polyubiquitinylated conjugates monoclonal antibody (clone FK2) (HRP conjugate) (BML-PW0150; Enzo Life Sciences), anti-PCNA/Pol30 (ab221196; Abcam).

Techniques: In Vivo, Mutagenesis, Conjugation Assay, Activity Assay, Expressing

Journal: iScience

Article Title: Methylated histones on mitotic chromosomes promote topoisomerase IIα function for high fidelity chromosome segregation

doi: 10.1016/j.isci.2023.106743

Figure Lengend Snippet:

Article Snippet: Mouse anti-Histone H2B , Cell Signaling Technology , Cat #2934 RRID: AB_2295301.

Techniques: Recombinant, Mutagenesis, Software, CRISPR