Journal: Journal of Biological Chemistry

Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex

doi: 10.1074/jbc.ra118.006290

Figure Lengend Snippet: Figure 4. Nrf1 interacts with the TIP60 complex. (A) Wild-type (WT) and Nrf1-/- NIH-3T3 cell lines were treated with 200 nM carfilzomib (CFZ) for 8 hours. The cells were then subjected to chromatin immunoprecipitation (ChIP) with one of IgG, Nrf1, RUVBL1, or TIP60 antibodies. These samples were then analyzed by quantitative PCR with primers specific for antioxidant response element (ARE)- containing promoter regions of proteasome genes PSMA7, PSMB7, and PSMD12. Error bars denote SD (n=3). (B) HEK293 cells stably expressing tagged Nrf1 (Nrf13xFLAG) were treated or not with 200 nM CFZ for 8 hours. The cell lysates were then subjected to immunoprecipitation with anti-FLAG beads and analyzed by immunoblotting with antibodies specific for FLAG, RUVBL1, and RUVBL2. The lysate lanes were loaded with 5% of the input that was used for immunoprecipitation. The experiments were performed three independent times and a representative blot is shown.

Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), Ubiquitin (1:3000), cleaved caspase-3 (1:3000) (all from Cell Signaling), INO80 (1:500; a gift from Dr. Landry (45)), PIH1 (1:1000), (Proteintech) and TIP60 (1:1500) (Abcam) and -Actin (1:10,000) (SigmaAldrich).

Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, Immunoprecipitation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Assembly of mTORC3 Involves Binding of ETV7 to Two Separate Sequences in the mTOR Kinase Domain

doi: 10.3390/ijms251810042

Figure Lengend Snippet: In vitro binding of the LBE sequence of the mTOR kinase domain to ETV7. ( A ). Top drawing: schematic representation of the N- and C-terminally extended LBE (highlighted in blue)fragment. The numbers indicate the number of amino acids in each of the LBE segments. Below is a FLAG immunoblot of an IP of the 120aa LBE fragment after overnight binding to ETV7 in vitro. Control (CTRL) shows the binding of human RUVBL2 to ETV7. ( B ). Left panel, schematic representation of the different deletions of the N- and C-terminal extended LBE fragment (120 amino acids) and underneath the amino acid sequence of the LBE domain with the amino acids essential for ETV7 binding in red; Right, FLAG immunoblot of an ETV7 IP of the different LBE fragments after overnight binding to ETV7 in vitro. Lanes containing fragments that lost binding to ETV7 are boxed in red. ( C ). FRB and LBE bind to ETV7 simultaneously. FLAG immunoblot of an ETV7 IP of LBE (120 amino acids) and Ndel-FRB fragments (ETV7:LBE:Ndel-FRB = 1:1:1, 1:1:5, 1:5:1) after overnight binding to ETV7 in vitro. ( D ). Binding of Ndel-FRB to ETV7 fragments containing the PNT domain and binding of LBE (120 amino acids) to ETV7 fragments containing the PNT and ETS domains in vitro.

Article Snippet: ETV7 protein (Cat #TP307742) and RUVBL2 (Cat #TP300933) were purchased from Origene (Rockville, MD, USA).

Techniques: In Vitro, Binding Assay, Sequencing, Western Blot, Control

Journal: bioRxiv

Article Title: Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies to self-antigens

doi: 10.1101/2023.03.20.533453

Figure Lengend Snippet: ( A ) Proteins that were immunoprecipitated by antibody 8-3 from lysates of MiaPaca2 cells were separated by SDS-PAGE and visualized by silver staining. Two bands were identified by mass spectrometry (red arrowheads). ( B ) MiaPaca2 cells were co-stained with 8-3, phalloidin, and an antibody specific for MYH10 after 30min treatment of the cells with DMSO or cytochalasin D. ( C ) Dot-blot assay is shown the reaction with F-actin or G-actin of 8-3, isotype control IgG, and a commercial actin specific antibody. ( D ) The binding of incremental concentrations of 8-3 to F-actin was measured by ELISA. ( E ) Proteins that were immunoprecipitated by recombinant antibody 19-3 from lysates of MiaPaca2 cells were separated by SDS-PAGE and visualized by silver staining. The proteins, RUVBL1 and RUVBL2, were identified by mass spectrometry. ( F ) MiaPaca2 cell were co-stained with 19-3 and an antibody specific for RUVBL2. ( G ) The binding of incremental concentrations of 19-3 to RUVBL2 was measured by ELISA. ( H ) Western blot analysis of recombinant antibody 15-7 detection of cytoplasmic proteins (Cyto) and mitochondria-enriched proteins (Mito) is shown. ( I ) Immunofluorescent staining of MiaPaca2 cells by 15-7 and an antibody specific for HSPD1 is shown. ( J ) The binding of incremental concentrations of 15-7 to HSPD1 was measured by ELISA. ( K ) The serum IgG titers of normal individuals and PDAC patients to F-actin (sera diluted 1:100), to RUVBL2 (sera diluted 1:1000), and to HSPD1(sera diluted 1:1000), respectively, were measured by ELISA. The non-parametric t-test was used for comparison. Scale bars in B , F and I are 50um.

Article Snippet: Other antibodies and dyes used were: Phalloidin (Thermo Fisher, #A12379), MYH10 (Atlas Antibodies, #HPA047541), cytochalasin D (Thermo Fisher, #PHZ1063), ACTIN (Cell Signaling Technology, #3700S), RUVBL2 (Atlas Antibodies, #HPA067966), RUVBL1 (Atlas Antibodies, # HPA019947), HSPD1 (Atlas Antibody, #HPA050025), HSPA9 (Atlas Antibody, #HPA000898), COX4I1 (Atlas antibody, #HPA002485), KRT19 (Abcam, #ab203445), human IgG isotype controls (Biolegend, #403502 and #403702).

Techniques: Immunoprecipitation, SDS Page, Silver Staining, Mass Spectrometry, Staining, Dot Blot, Control, Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Western Blot, Comparison

Journal: bioRxiv

Article Title: Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies to self-antigens

doi: 10.1101/2023.03.20.533453

Figure Lengend Snippet: ( A ) 8-3 staining after siRNA knockdown of ACTB in MiaPaca2 cells, co-stained with phalloidin, MYH10 antibody and DAPI. ( B ) Measurement of 19-3 binding to recombinant RUVBL1 and RUBL2 proteins by ELISA. Positive controls using polyclonal rabbit antibodies anti-RUVBL1 (a-RUVBL1) or anti-RUVBL2 (a-RUVBL2). ( C ) 19-3 staining after siRNA knockdown of RUVBL2 in MiaPaca2 cells, co-stained with anti-RUVBL2 rabbit antibody and DAPI. ( D ) Co-staining of 15-7 with mitochondria markers COX4I1, HSPA9 and HSPD1 in MiaPaca2 cells. Scale bars in A, C and D are 50um.

Article Snippet: Other antibodies and dyes used were: Phalloidin (Thermo Fisher, #A12379), MYH10 (Atlas Antibodies, #HPA047541), cytochalasin D (Thermo Fisher, #PHZ1063), ACTIN (Cell Signaling Technology, #3700S), RUVBL2 (Atlas Antibodies, #HPA067966), RUVBL1 (Atlas Antibodies, # HPA019947), HSPD1 (Atlas Antibody, #HPA050025), HSPA9 (Atlas Antibody, #HPA000898), COX4I1 (Atlas antibody, #HPA002485), KRT19 (Abcam, #ab203445), human IgG isotype controls (Biolegend, #403502 and #403702).

Techniques: Staining, Knockdown, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies to self-antigens

doi: 10.1101/2023.03.20.533453

Figure Lengend Snippet: ( A ) Comparison of IgG titer to F-actin, RUVBL2 and HSPD1 of plasma samples diluted at 1/100 or 1/1000 by ELISA. Background signals from secondary antibody only are shown in a dashed line. ( B ) Comparison of IgG response to F-actin, RUVBL2 and HSPD1 between naïve treated (naive) and neoadjuvant treated (NAT) PDAC patients. The non-parametric t-test is used for comparisons.

Article Snippet: Other antibodies and dyes used were: Phalloidin (Thermo Fisher, #A12379), MYH10 (Atlas Antibodies, #HPA047541), cytochalasin D (Thermo Fisher, #PHZ1063), ACTIN (Cell Signaling Technology, #3700S), RUVBL2 (Atlas Antibodies, #HPA067966), RUVBL1 (Atlas Antibodies, # HPA019947), HSPD1 (Atlas Antibody, #HPA050025), HSPA9 (Atlas Antibody, #HPA000898), COX4I1 (Atlas antibody, #HPA002485), KRT19 (Abcam, #ab203445), human IgG isotype controls (Biolegend, #403502 and #403702).

Techniques: Comparison, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: Structure of Yin Yang 1 Oligomers That Cooperate with RuvBL1-RuvBL2 ATPases

doi: 10.1074/jbc.m114.567040

Figure Lengend Snippet: FIGURE 6. Analysis of the interaction of purified YY1 with RuvBL1 and RuvBL2. A, SDS-PAGE and SimplyBlue (Invitrogen) staining of purified His-RuvBL1, His-RuvBL2, His-RuvBL1-RuvBL2, and RuvBL1-RuvBL2. B, chromatograms and SDS-PAGE analysis of the fractions from a SEC of His-RuvBL1, His-RuvBL2, and His-RuvBL1-RuvBL2 (solid line) and RuvBL1-RuvBL2 (dash line) using a Superdex 200 PC 3.2/30 (GE Healthcare) column. Proteins were stained using SimplyBlue (Invitrogen)(His-RuvBL1,His-RuvBL1-RuvBL2,andRuvBL1-RuvBL2)orbysilverstaining(His-RuvBL2).Thepositionsforthedifferentoligomericspecies(12-mer, dodecamer; 6-mer, hexamer; 1-mer, monomer) are indicated in each case and were determined by comparison with molecular weight standards. The asterisk (*) in the case of His-RuvBL1 SEC indicates a peak of aggregated material as observed by EM. C, pulldown experiments of Strep-II-YY1 and His-RuvBL1 or His-RuvBL2. Strep-II-YY1 was incubated without (lane 1) or with His-RuvBL1 (lane 3) or His-RuvBL2 (lane 5) and affinity purified using the Strep-II-tag present in YY1. As a control, His-RuvBL1 (lane 2) or His-RuvBL2 (lane 4) was purified in the same conditions but in the absence of YY1. D, pulldown experiments of Strep-II-YY1andHis-RuvBL1-RuvBL2orRuvBL1-RuvBL2.Strep-II-YY1wasincubatedwithout(lane1)orwithHis-RuvBL1-RuvBL2(lane3)orRuvBL1-RuvBL2(lane 5) and affinity-purified as in A. As a control, His-RuvBL1-RuvBL2 (lane 2) or RuvBL1-RuvBL2 (lane 4) was pull downed in the same conditions as those before but in the absence of YY1.

Article Snippet: The RuvBL2 complete human ORF cDNA (accession number NM_006666) was purchased from Origene as GFP-tagged transfection-ready DNA.

Techniques: Purification, SDS Page, Staining, Comparison, Molecular Weight, Incubation, Affinity Purification, Control

Journal: Journal of Biological Chemistry

Article Title: Structure of Yin Yang 1 Oligomers That Cooperate with RuvBL1-RuvBL2 ATPases

doi: 10.1074/jbc.m114.567040

Figure Lengend Snippet: FIGURE 7. YY1 and RuvBL1-RuvBL2 cooperate in DNA binding. A, left panel, EMSA assays showing binding of Strep-II-YY1 to several DNA substrates (described under “Experimental Procedures”) and represented as a schematic in each gel. Binding reactions contained 0, 240, 120, 60, or 30 nM protein and 0.3 nM DNA species: HJ (J3) (lanes 1–5), dsDNA non-consensus sequence (dsDNA_no_sp_1) (lanes 6–10), 80-nt ssDNA (lanes 11–14), and dsDNA consensus sequence (dsDNA_sp_1) (lanes 15–19). Right panel, EMSA assays showing binding of His-RuvBL1-RuvBL2 to the same DNA substrates. Binding reactions contained 0, 1.4, 0.7, 0.35, or 0.17 M protein and 0.3 nM DNA species: HJ (J3) (lanes 1–5), dsDNA non-consensus sequence (dsDNA_no_sp_1) (lanes 6–10), 80-nt ssDNA (lanes 11–14), and dsDNA consensus sequence dsDNA_sp_1) (lanes 15–19). Reactions were performed as described under “Experimental Procedures,” and protein-DNA complexes were visualized by 6% PAGE and autoradiography. B, RuvBL1-RuvBL2 enhances the binding of YY1 to dsDNA either containing (right panel, dsDNA_sp_1) or not the consensus sequence (left panel, dsDNA_no_sp_1). Reactions assembled on ice contained combinations of a decreasing concentration of His-RuvBL1-RuvBL2 (1.4, 0.7, 0.35 or 0.17 M) and a fixed concentration of Strep-II-YY1 (120 nM in the left panel and 15 nM in the right panel). After DNA addition (0.3 nM), samples were incubated for 30 min at 37 °C before electrophoresis. C, YY1 and RuvBL1-RuvBL2 also cooperate in ssDNA binding. Shown is a similar experiment as B but using a 80-nt ssDNA (0.3 nM) (lanes 1–9). His-RuvBL1-RuvBL2 concentrations were varied as indicated from 700 to 87 nM, and the fixed concentration of Strep-II-YY1 used was 120 nM. D, EMSA of the enhancement in binding of Strep-II-YY1 to HJ (J3) (0.3 nM) in the presence of the indicated concentrations His-RuvBL1-RuvBL2 (concentrations are expressed in nM).

Article Snippet: The RuvBL2 complete human ORF cDNA (accession number NM_006666) was purchased from Origene as GFP-tagged transfection-ready DNA.

Techniques: Binding Assay, Sequencing, Autoradiography, Concentration Assay, Incubation, Electrophoresis

Journal: Journal of Biological Chemistry

Article Title: Structure of Yin Yang 1 Oligomers That Cooperate with RuvBL1-RuvBL2 ATPases

doi: 10.1074/jbc.m114.567040

Figure Lengend Snippet: FIGURE8.YY1andRuvBL2functionduringG2topromoteRAD51fociformation,butnotH2AXorRPAfoci,afterexposuretoIR.A,A549cellsweretransfected withscrambledsiRNA(siCTR)orsiRNAdirectedagainstYY1,RuvBL2,orBRCA2,irradiated(3Gy)andharvested2or8hlaterandimmunostainedforH2AXandCENPF (to identify G2 phase cells). Average H2AX foci per G2 cell were quantified (right panel). B, cells transfected as in A were irradiated (3 Gy), harvested after 2 h, and immunostained for RPA and CENPF. Average RPA foci per G2 cell were quantified (right panel). C, cells transfected as inA were immunostained for RAD51 and CENPF, and average RAD51 foci per G2 cell were quantified (right panel). All data (A–C) are the means of 3 experiments; error bars, S.D.

Article Snippet: The RuvBL2 complete human ORF cDNA (accession number NM_006666) was purchased from Origene as GFP-tagged transfection-ready DNA.

Techniques: Irradiation, Transfection

Journal: Journal of Biological Chemistry

Article Title: Structure of Yin Yang 1 Oligomers That Cooperate with RuvBL1-RuvBL2 ATPases

doi: 10.1074/jbc.m114.567040

Figure Lengend Snippet: FIGURE 9. RuvBL2 cooperates with YY1 to promote RAD51 foci formation, and the ATPase activity of RuvBL2 is required for this activity. A, A549 cells were transfectedwithscrambledsiRNA(siCTR)orsiRNAsdirectedagainstYY1,RuvBL2,orBRCA2,asindicated,irradiated(3Gy),harvested2or8hlater,andimmunostained for H2AX and CENPF (to identify G2 phase cells). Average H2AX foci per G2 cell were quantified (right panel). B, cells treated as in A were immunostained for RAD51 and CENPF, and average RAD51 foci per G2 cell were quantified (right panel). All data (A and B) are the mean of 3 experiments; error bars, S.D. C, top, Western blot analysisofwholecellextractspreparedfromA549cellstransfectedwiththeindicatedsiRNA.Bottom,WesternblotanalysisofwholecellextractspreparedfromA549 cells transfected with single or double siRNA constructs as indicated. Anti-KAP1 is shown as a loading control. D, U2OS cells transfected with siRNA directed against RuvBL2 and either GFP, GFP-RuvBL2, or GFP-RuvBL2-K83A were irradiated (3 Gy), harvested, and immunostained for RAD51 and CENPF. Average RAD51 foci per GFP positive G2 cell were quantified and are presented as the means of three experiments; error bars, S.D.

Article Snippet: The RuvBL2 complete human ORF cDNA (accession number NM_006666) was purchased from Origene as GFP-tagged transfection-ready DNA.

Techniques: Activity Assay, Irradiation, Western Blot, Transfection, Construct, Control