ruvbl2  (OriGene)

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: The 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: 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, and RuvBL1-RuvBL2) or by silver staining (His-RuvBL2). The positions for the different oligomeric species (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-YY1 and His-RuvBL1-RuvBL2 or RuvBL1-RuvBL2. Strep-II-YY1 was incubated without (lane 1) or with His-RuvBL1-RuvBL2 (lane 3) or RuvBL1-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 {"type":"entrez-nucleotide","attrs":{"text":"NM_006666","term_id":"1007385611","term_text":"NM_006666"}} NM_006666 ) was purchased from Origene as GFP-tagged transfection-ready DNA.

Techniques: Purification, SDS Page, Staining, Silver Staining, Molecular Weight, Incubation, Affinity Purification

Journal: The 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: 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 μm 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 μm) 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 {"type":"entrez-nucleotide","attrs":{"text":"NM_006666","term_id":"1007385611","term_text":"NM_006666"}} NM_006666 ) was purchased from Origene as GFP-tagged transfection-ready DNA.

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

Journal: The 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: DNA binding affinities of YY1 and RuvBL1-RuvBL2

Article Snippet: The RuvBL2 complete human ORF cDNA (accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_006666","term_id":"1007385611","term_text":"NM_006666"}} NM_006666 ) was purchased from Origene as GFP-tagged transfection-ready DNA.

Techniques: Binding Assay

Journal: The 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: YY1 and RuvBL2 function during G2 to promote RAD51 foci formation, but not γH2AX or RPA foci, after exposure to IR. A, A549 cells were transfected with scrambled siRNA (siCTR) or siRNA directed against YY1, RuvBL2, or BRCA2, irradiated (3 Gy) and harvested 2 or 8 h later and immunostained for γH2AX and CENPF (to identify G2 phase cells). Average γH2AX 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 in A 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 {"type":"entrez-nucleotide","attrs":{"text":"NM_006666","term_id":"1007385611","term_text":"NM_006666"}} NM_006666 ) was purchased from Origene as GFP-tagged transfection-ready DNA.

Techniques: Transfection, Irradiation

Journal: The 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: RuvBL2 cooperates with YY1 to promote RAD51 foci formation, and the ATPase activity of RuvBL2 is required for this activity. A, A549 cells were transfected with scrambled siRNA (siCTR) or siRNAs directed against YY1, RuvBL2, or BRCA2, as indicated, irradiated (3 Gy), harvested 2 or 8 h later, and immunostained for γH2AX and CENPF (to identify G2 phase cells). Average γH2AX 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 analysis of whole cell extracts prepared from A549 cells transfected with the indicated siRNA. Bottom, Western blot analysis of whole cell extracts prepared from A549 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 {"type":"entrez-nucleotide","attrs":{"text":"NM_006666","term_id":"1007385611","term_text":"NM_006666"}} NM_006666 ) was purchased from Origene as GFP-tagged transfection-ready DNA.

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