Journal: Journal of Biological Chemistry

Article Title: FBXO3 Protein Promotes Ubiquitylation and Transcriptional Activity of AIRE (Autoimmune Regulator)

doi: 10.1074/jbc.m116.724401

Figure Lengend Snippet: FIGURE 8. Ubiquitylation of AIRE increases the binding and recruitment of P-TEFb to target genes. A, mutations of Thr-68 and Ser-156 influence the AIRE interaction with P-TEFb. 293T cells expressed WT mHis:AIRE or mutant mHis:AIRE proteins for 48 h. Whole cell lysates were immunoprecipitated (IP) with anti-His antibodies followed by Western blotting with indicated antibodies. B, knockdown of FBXO3 decreases interactions between AIRE and P-TEFb. f:AIRE was expressed in 293T cells with control (siScr) or siFBXO3 (siFBXO3) RNAs for 72 h before harvesting. Total cell lysates were incubated with anti-FLAG beads. AIRE, the endogenous CycT1 and FBXO3 proteins, were examined with indicated antibodies. The top panels contain immunoprecipitated CycT1 and f:AIRE proteins. The lower panels contain 3% input CycT1, f:AIRE, and FBXO3 proteins. C and D, interactions between AIRE and P-TEFb on DNA are decreased by deleterious mutations in AIRE and the depletion of FBXO3. C, presented are ChIPs of AIRE and CycT1 at promoters of TSA genes (KRT14 and S100A8) in cells expressing WT and mutant mHis:AIRE proteins. D, presented are ChIPs of AIRE and CycT1 at the promoter of TSA genes (KRT14 and S100A8) in control or FBXO3-depleted cells. Bars represent the S.E. of three independent experiments (S.E., n 3). Student’s t test was preformed to measure the significance of the data (*, p 0.05; **, p 0.01).

Article Snippet: Antibodies used for Western blotting, co-immunoprecipitation, or immunofluorescence were: AIRE (H300, sc-33188, and D17, sc-17986, Santa Cruz), AIRE (phospho Ser-156) antibody (ab78211, Abcam), Cullin 1 (D-5, sc-17775, Santa Cruz), c-Myc (9E10, HRP, ab62928, Abcam), CycT1 (H-245, sc-10750, Santa Cruz), FBXO3 (H300, sc-134722, Santa Cruz), FLAG M2-peroxidase (A8592, HRP, Sigma), FLAG M2-agarose from mouse (A2220, Sigma), GAPDH (6C5, sc-32233, Santa Cruz), His (H3, sc-8036, Santa Cruz), HA (Y-11, sc-805, Santa Cruz), RBX1 (34 –2500, Invitrogen), UBC4 (Y20, sc-100617, Santa Cruz), ubiquitin (P4D1, sc-8017, Santa Cruz), and V5-HRP antibodies (R961–25, Life Technologies) as well as rabbit and mouse control IgG (Santa Cruz).

Techniques: Binding Assay, Mutagenesis, Immunoprecipitation, Western Blot, Knockdown, Control, Incubation, Expressing

Journal: Journal of Virology

Article Title: Rotavirus NSP1 Associates with Components of the Cullin RING Ligase Family of E3 Ubiquitin Ligases

doi: 10.1128/jvi.00704-16

Figure Lengend Snippet: Figure 3. HaloTag-NSP1 proteins associate with dominant negative (DN) Cul3 and Cul1 880

Article Snippet: Rabbit polyclonal antibodies to Halo (Promega, 162 G9281), IRF3 (Cell Signaling, cs-11904), β-TrCP (Cell Signaling, cs-4394), Cul1 (Novus, NB-163 100-91724), Cul3 (Novus, NB100-58788), MBP (Santa Cruz Biotechnology, sc-808), and PCNA 164 (Santa Cruz Biotechnology, sc-7907) were used at a 1:1,000 dilution.

Techniques: Dominant Negative Mutation

Journal: Antioxidants

Article Title: The Antioxidant Enzyme Methionine Sulfoxide Reductase A (MsrA) Interacts with Jab1/CSN5 and Regulates Its Function

doi: 10.3390/antiox9050452

Figure Lengend Snippet: In vivo regulation of Jab1 activity by MsrA through monitoring Cul-1 neddylation levels in mouse brain and liver extracts. ( A ), a–d. Mouse extracts from 6 months old mice ( n = 3) were made in PBS and in the presence of protease inhibitors cocktail (Sigma-Aldrich). Immunoprecipitation (IP) experiments were performed on brain and liver extracts (500 µg of protein per extract) using either anti-Cul-1 antibody or anti-Nedd8 antibody followed by Western blot (WB) analyses using anti-Nedd8 antibody or anti-Cul-1 antibody as the primary antibody, respectively. Ae. In a unique experiment, Jab1 was used as a “fishing” probe in an IP experiment using brain extracts, followed by Western blot analysis using anti-Cul-1antibody. Only the 100-kDa neddylated Cul-1 protein was identified due to the specific pull-down of the neddylated form of Cul-1 by Jab1. ( B ) Quantification of each band identified by Western blot analyses described in Panel ( A ) a–e, as determined by using the NIH Image-J program. All the observed differences in the observed band levels between the WT and MT pairs were statistically significant as judged by student t -test analysis (* p < 0.01, n = 3 per strain). ( C ) Loading controls for the liver and brain protein levels, following Coomassie blue staining (to confirm the use of equal amount of proteins from each mouse strain per tissue in the IP experiments. WT, wild type; M, MsrA KO. ( D ) Western blot analysis of the mouse brain extracts using anti Jab1 antibody as the primary antibody and quantified by the NIH Image-J program. WT, wild type; MT, MsrA KO. Arrows shown in ( A ) a–e indicate the position of the neddylated 100-kDa Cul-1 (deneddylated form runs as ~90 kDa protein, not detected). The shown WB and Coomassie blue staining experiments are representatives of three independent experiments.

Article Snippet: To achieve this goal, IP experiments were performed using the anti-Cul-1 antibody (Novus, Littleton, CO, USA) as the IP antibody and the anti-Nedd8 (Boster Bio, Pleasanton, CA, USA) as the primary antibody in the subsequent Western blot analysis and vice-versa.

Techniques: In Vivo, Activity Assay, Immunoprecipitation, Western Blot, Staining

Journal: The Journal of Biological Chemistry

Article Title: The Human COP9 Signalosome Protects Ubiquitin-conjugating Enzyme 3 (UBC3/Cdc34) from ?-Transducin Repeat-containing Protein (?TrCP)-mediated Degradation *

doi: 10.1074/jbc.M109.076661

Figure Lengend Snippet: UBC3 is a target of SCFβTrCP. A, UBC3 protein levels in CSN knockdown cells are rescued by CUL1 suppression. shRNA-transduced 293T cells were transfected with a control oligonucleotide or with two different siRNAs targeting CUL1. UBC3 content in total lysates was analyzed by Western blotting (upper panel). Cullin mRNA levels were analyzed by quantitative reverse transcription-PCR (lower panel). B, endogenous UBC3 and βTrCP interact. UBC3 (middle panel) and βTrCP (right panel) were immunoprecipitated (IP) from HEK293T cell lysates with specific antibodies. The control used was the irrelevant mouse (left panel) or rabbit (right panel) IgG antibody. WB, Western blot. C, the F-box is not required for βTrCP interaction with UBC3. 293T cells were transfected with the indicated vectors (full-length βTrCP (βTrCP-FL) and the F-box deletion mutant βTrCPΔFbox), and βTrCP was immunoprecipitated with anti-FLAG antibodies. UBC3 wt, wild-type UBC3. D, βTrCP overexpression promotes UBC3 ubiquitination. 293T cells were transfected as indicated. HA-UBC3 was immunoprecipitated with anti-HA antibody, and ubiquitinated forms of UBC3 were detected by anti-Myc Western blotting. E, down-regulation of βTrCP increases UBC3 protein levels. 293T cells were transfected with two different siRNA oligonucleotides that target both βTrCP1 and βTrCP2. F, down-regulation of SKP2 has no effect on UBC3 protein levels. 293T cells were transfected with two siRNA oligonucleotides targeting SKP2.

Article Snippet: The following antibodies were used for this study: anti-CSN5 monoclonal antibody SP282, described previously ( 29 ); anti-CSN4 antibody (A300-013A) from Bethyl Laboratories; anti-Cul1 (71-8700) and anti-p45 SKP2 (32-3300) antibodies from Zymed Laboratories Inc.; anti-p27 (C-19, sc-528), IκB-α (C-21, sc-371), UBC4 (C-15, sc-15000), and UBC9 (H-81, sc-10759) antibodies from Santa Cruz Biotechnology; anti-β-catenin antibody ( {"type":"entrez-nucleotide","attrs":{"text":"C19220","term_id":"1631491","term_text":"C19220"}} C19220 ) from Transduction Laboratories; anti-UBC3 polyclonal antibody (4997) from Cell Signaling; anti-UBC3 monoclonal antibody (90.12) from GeneTex, Inc.; anti-hemagglutinin (HA) (clone HA-7, H9658), anti-β-tubulin (clone TUB2.1, T4026), anti-actin (20-33, A5060), anti-Myc monoclonal (clone 9E10), and anti-FLAG M2 antibodies from Sigma; and anti-USP15 antibody from Abnova.

Techniques: shRNA, Transfection, Western Blot, Immunoprecipitation, Mutagenesis, Over Expression

Journal: Molecular cell

Article Title: PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network.

doi: 10.1016/j.molcel.2019.12.013

Figure Lengend Snippet: Figure 1. A Toolbox to Monitor Endogenous CRL4 Complexes Schematic representation of the PIKES approach: (1) 293T/17 cells were genetically engineered to carry a 3xFLAG or 3xHA on Cul4A or Cul4B or both to enable rapid and efficient (2) immunoprecipitation of endogenous CRL4 assemblages. (3) Endogenous CRL4 ligase complexes were then evaluated systematically using global and targeted proteomic approaches. First, the protein interaction network of interest was analyzed at endogenous protein levels, followed by a kinetic characterization of interactions, and the development of chemical and biochemical methods to control and preserve the composition of protein complexes. Subsequently, the cellular concentrations and complex stoichiometries were analyzed using QconCAT standards, and induced changes in complex composition were monitored quantitatively. See also Figures S1, S7, and S8.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-Cul4A (epitope Cell Signaling Technology 2699S, RRID:AB_2086563 Rabbit polyclonal anti-Cul4B (epitope AA Bethyl A303-863A, RRID:AB_2620214 Rabbit polyclonal anti-Cul4B (epitope AA Sigma-Aldrich C9995, RRID:AB_1840781 Mouse monoclonal anti-Cul1 (D-5) Santa Cruz Biotechnology Sc-17775, RRID:AB_627325 Rabbit polyclonal anti-Cand1 Bethyl A302-901A, RRID:AB_10663486 Rabbit monoclonal anti-DDB2 Abcam ab181136 Rabbit monoclonal anti-DDB1 Abcam ab109027, RRID:AB_10859111 Rabbit polyclonal anti-pCHK-1 Cell Signaling Technology 2341S, RRID:AB_330023 Rabbit polyclonal anti-pH2A.x Cell Signaling Technology 2577S, RRID:AB_2118010 Rabbit polyclonal anti-CRBN Sigma-Aldrich HPA045910-100UL, RRID:AB_10960409 Rabbit polyclonal anti-CSN5 Santa Cruz Biotechnology Sc-393725 Rat monoclonal anti-HA-HRP (clone 3F10) Roche 34071100 Mouse monoclonal anti-FLAG Sigma-Aldrich A8592-2MG, RRID:AB_439702 Mouse monoclonal anti-GAPDH Santa Cruz Biotechnology sc-365062, RRID:AB_10847862 Rabbit monoclonal anti-BRD4 Abcam ab128874, RRID:AB_11145462 Rabbit monoclonal anti-CK1alpha Abcam ab206662 Rabbit polyclonal anti-ZFP91 Bethyl A303-245A, RRID:AB_10953803 Rabbit polyclonal anti-RBM39 Bethyl A300-291A, RRID:AB_263411 Mouse monoclonlal anti-b-actin Sigma-Aldrich A5316, RRID:AB_476743 Rabbit monoclonal anti-GFP Abcam ab32146, RRID:AB_732717 Rabbit TrueBlot Anti-Rabbit IgG HRP Rockland 18-8816-33, RRID:AB_2610848 Donkey-anti-Mouse-HRP secondary antibody Jackson Laboratories 715-035-151, RRID:AB_2340771 Donkey anti-Rabbit-HRP secondary antibody Jackson Laboratories 711-035-152, RRID:AB_10015282 IRDye 800CW Donkey-anti-Mouse secondary antibody Li-COR 926-32212, RRID:AB_621847 IRDye 680RD Donkey-anti-Rabbit secondary antibody Li-COR 926-68073, RRID:AB_10954442 EZview Red Anti-HA Affinity Gel Sigma-Aldrich E6779-1ML, RRID:AB_10109562 EZview Red Anti-FLAG Affinity Gel Sigma-Aldrich F2426-1ML, RRID:AB_2616449 EZview Red GSH Affinity Gel Sigma-Aldrich E6406-1ML Chemicals, Peptides, and Recombinant Proteins CRL4 SpikeMix This study, synthesized by JPT Peptide Technologies, Berlin, Germany N/A CRL4 QconCAT This study, synthesized by PolyQuant GmbH, Bad Abbach, Germany N/A LysC protease Wako 125-02543 Trypsin protease Promega V511C MLN4924 Calbiochem 5.05477.0001 CSN5i-3 Custom synthesis N/A MLN7243 MedKoo Biosciences 206163 Bortezomib Selleck Chemicals S1013 CB5083 Custom synthesis N/A Indisulam MedKoo Biosciences 201540 Lenalidomide Selleck Chemicals S1029 (Continued on next page) e1 Molecular Cell 77, 1–15.e1–e9, March 5, 2020

Techniques: Immunoprecipitation, Control

Journal: Molecular cell

Article Title: PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network.

doi: 10.1016/j.molcel.2019.12.013

Figure Lengend Snippet: Figure 3. Individual CRL4 Ligases Show Cullin-Scaffold Preferences and Differ up to 100-Fold in Absolute Abundance (A) Schematic of CRL4 QconCAT experiments to determine cellular concentrations and complex stoichiometries. (B–D) Absolute quantification of CRL4 components in whole cell lysates as well as immunoprecipitated Cul4 complexes. *Cul4 scaffolds were measured by targeting peptides common (cP) or unique (uP) to Cul4A and Cul4B. (E) Summary of cellular organization of CRL4 complexes. (F) and (G) Cellular concentrations of assembled CRL4s as well as DCAFs. (H) Correlation plot of percentage assembled (percentage of DCAF Cul4-bound versus free) and CRL4 ligase abundance (percentage of individual Cul4DCAF

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-Cul4A (epitope Cell Signaling Technology 2699S, RRID:AB_2086563 Rabbit polyclonal anti-Cul4B (epitope AA Bethyl A303-863A, RRID:AB_2620214 Rabbit polyclonal anti-Cul4B (epitope AA Sigma-Aldrich C9995, RRID:AB_1840781 Mouse monoclonal anti-Cul1 (D-5) Santa Cruz Biotechnology Sc-17775, RRID:AB_627325 Rabbit polyclonal anti-Cand1 Bethyl A302-901A, RRID:AB_10663486 Rabbit monoclonal anti-DDB2 Abcam ab181136 Rabbit monoclonal anti-DDB1 Abcam ab109027, RRID:AB_10859111 Rabbit polyclonal anti-pCHK-1 Cell Signaling Technology 2341S, RRID:AB_330023 Rabbit polyclonal anti-pH2A.x Cell Signaling Technology 2577S, RRID:AB_2118010 Rabbit polyclonal anti-CRBN Sigma-Aldrich HPA045910-100UL, RRID:AB_10960409 Rabbit polyclonal anti-CSN5 Santa Cruz Biotechnology Sc-393725 Rat monoclonal anti-HA-HRP (clone 3F10) Roche 34071100 Mouse monoclonal anti-FLAG Sigma-Aldrich A8592-2MG, RRID:AB_439702 Mouse monoclonal anti-GAPDH Santa Cruz Biotechnology sc-365062, RRID:AB_10847862 Rabbit monoclonal anti-BRD4 Abcam ab128874, RRID:AB_11145462 Rabbit monoclonal anti-CK1alpha Abcam ab206662 Rabbit polyclonal anti-ZFP91 Bethyl A303-245A, RRID:AB_10953803 Rabbit polyclonal anti-RBM39 Bethyl A300-291A, RRID:AB_263411 Mouse monoclonlal anti-b-actin Sigma-Aldrich A5316, RRID:AB_476743 Rabbit monoclonal anti-GFP Abcam ab32146, RRID:AB_732717 Rabbit TrueBlot Anti-Rabbit IgG HRP Rockland 18-8816-33, RRID:AB_2610848 Donkey-anti-Mouse-HRP secondary antibody Jackson Laboratories 715-035-151, RRID:AB_2340771 Donkey anti-Rabbit-HRP secondary antibody Jackson Laboratories 711-035-152, RRID:AB_10015282 IRDye 800CW Donkey-anti-Mouse secondary antibody Li-COR 926-32212, RRID:AB_621847 IRDye 680RD Donkey-anti-Rabbit secondary antibody Li-COR 926-68073, RRID:AB_10954442 EZview Red Anti-HA Affinity Gel Sigma-Aldrich E6779-1ML, RRID:AB_10109562 EZview Red Anti-FLAG Affinity Gel Sigma-Aldrich F2426-1ML, RRID:AB_2616449 EZview Red GSH Affinity Gel Sigma-Aldrich E6406-1ML Chemicals, Peptides, and Recombinant Proteins CRL4 SpikeMix This study, synthesized by JPT Peptide Technologies, Berlin, Germany N/A CRL4 QconCAT This study, synthesized by PolyQuant GmbH, Bad Abbach, Germany N/A LysC protease Wako 125-02543 Trypsin protease Promega V511C MLN4924 Calbiochem 5.05477.0001 CSN5i-3 Custom synthesis N/A MLN7243 MedKoo Biosciences 206163 Bortezomib Selleck Chemicals S1013 CB5083 Custom synthesis N/A Indisulam MedKoo Biosciences 201540 Lenalidomide Selleck Chemicals S1029 (Continued on next page) e1 Molecular Cell 77, 1–15.e1–e9, March 5, 2020

Techniques: Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Multiple UBXN family members inhibit retrovirus and lentivirus production and canonical NFκΒ signaling by stabilizing IκBα

doi: 10.1371/journal.ppat.1006187

Figure Lengend Snippet: (A) FLAG-UBXN1 was co-transfected with Myc-Cul1, as indicated, and immunoprecipitated (IP) using anti-FLAG antibody, followed by immunoblotting (IB) using anti-FLAG or Myc antibody; β−tubulin served as loading control; (B) Left: schematic of Cul1 deletion constructs, with indicated regions that bind to Skp1 and Rbx1 (green and yellow, respectively, not precisely to scale); right: FLAG-Cul1 constructs were transfected into HEK293 cells, as indicated at top; endogenous UBXN1 was detected using anti-UBXN1 antibody and β−tubulin again served as loading control; (C) left: At top: Co-IP of endogenous UBXN1 and Cul1 from HFFs, after stimulating cells with 10 ng/mL TNFα; input of various proteins shown at bottom; right: relative band intensity of IκBα and Cul1, normalized against the density of β−tubulin (in the left immunoblot image); (D) left: Transfection of HEK293 cells with either empty vector or plasmid encoding FLAG-UBXN1, treated with 5 ng/mL TNFα for the indicated times, and cell lysates immunoblotted for all three indicated proteins, with β−tubulin serving as loading control; right: relative band intensity of IκBα normalized against the density of β−tubulin (in the left immunoblot image); (E) Similar to (D) except that NFκB-FFLUC reporter was co-transfected and cell lysates assayed for luciferase activity, normalized to co-transfected Renilla-LUC reporter, in the presence (grey bars) or absence (black bars) of UBXN1. Experiments of (C) and (D) were repeated at least twice, with similar results.

Article Snippet: Rabbit anti-Cul1 monoclonal antibody was from Novus Biologicals (NBP1-40523).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, Construct, Co-Immunoprecipitation Assay, Plasmid Preparation, Luciferase, Activity Assay

Journal: PLoS Pathogens

Article Title: Multiple UBXN family members inhibit retrovirus and lentivirus production and canonical NFκΒ signaling by stabilizing IκBα

doi: 10.1371/journal.ppat.1006187

Figure Lengend Snippet: (A) 293T cells were transiently transfected with the indicated FLAG-UBXN1 constructs or FLAG-Cul1 in the presence of either Myc-Rbx1 or Myc-Skp1 as indicated, with co-IP at top and input, including β-tubulin, at bottom; (B) Similar to (A) , except decreasing amounts of Myc-UBXN1 were transfected along with Myc-Skp1 and either empty vector (EV), ½-1 FLAG-Cul1, FLAG-Cul1, or 1/3-1 FLAG-Cul1, as indicated, with co-IP at top and input, including β-tubulin, at bottom; (C) Similar to (B) , except that Myc-Rbx1 was transfected along with EV, either 0-1/2 FLAG-Cul1, FLAG-Cul1, or 0-2/3 FLAG-Cul1, as indicated, with co-IP at top and input, including Cox IV, at bottom. Note that co-expression of Cul1 increased Rbx1 levels.

Article Snippet: Rabbit anti-Cul1 monoclonal antibody was from Novus Biologicals (NBP1-40523).

Techniques: Transfection, Construct, Co-Immunoprecipitation Assay, Plasmid Preparation, Expressing

Journal: PLoS Pathogens

Article Title: Multiple UBXN family members inhibit retrovirus and lentivirus production and canonical NFκΒ signaling by stabilizing IκBα

doi: 10.1371/journal.ppat.1006187

Figure Lengend Snippet: (A) Left: immunoblot of indicated proteins from HFFs stably transduced with either empty HIV-based vector or vector encoding anti-UBXN1 shRNA, after stimulation with 5 ng/mL TNFα for indicated times; right: relative band intensity of IκBα, normalized to β−tubulin (in the left immunoblot image); (B) Quantification of NFκB (left) and HIV LTR (right) FFLUC reporters in cell lines of (A) , normalized to co-transfected Renilla-LUC plasmid; for NFκB reporter cells were treated for 4 h with 5 ng/mL TNFα 48 h post-transfection; (C) Left: immunoblot of indicated proteins from UBXN1-/- HPRT-/- and control HPRT-/- MEFs, after stimulation with 10 ng/mL TNFα for indicated times (upper) or treated with 1 μM Bortezomib for 5 h and then stimulated with 10 ng/mL TNFα for the indicated times (lower); right: relative band intensity of IκBα, normalized to β−tubulin (in the left immunoblot images); (D) Confocal immunofluorescence microscopy of UBXN1 and Cul1 in UBXN1-/- HPRT-/- and control HPRT-/- MEFs. Nuclear DNA stained with TO-PRO-3 (I); UBXN1 (II) and Cul1 (III) were stained using secondary antibodies conjugated to Alexa Fluor 546 and 488, respectively; IV shows merge; Scale bar = 10 μm; (E) Quantification of NFκB and HIV LTR-FFLUC reporters in UBXN1-/- HPRT-/- and control HPRT-/- MEFs. Left: NFκB-FFLUC values 48 h post-transfection in the presence of 5 ng/mL TNFα for 4h, normalized to Renilla-LUC reporter; right: HIV LTR-FFLUC values 48 hrs post-transfection, similarly normalized; (F) JLAT10.6 T cells stably transduced with either HIV-based vector encoding anti-UBXN1 shRNA or control empty vector, after stimulating the cells with 5 ng/mL TNFα for the indicated times and measuring % eGFP+ by FACS. Data in (B) and (E) represent mean ± SEM (n = 3). ** p < 0.005, * p < 0.05 by student’s t-test. Experiments of (A) and (C) were repeated several times, with similar results.

Article Snippet: Rabbit anti-Cul1 monoclonal antibody was from Novus Biologicals (NBP1-40523).

Techniques: Western Blot, Stable Transfection, Transduction, Plasmid Preparation, shRNA, Transfection, Control, Immunofluorescence, Microscopy, Staining

Journal: PLoS Pathogens

Article Title: Multiple UBXN family members inhibit retrovirus and lentivirus production and canonical NFκΒ signaling by stabilizing IκBα

doi: 10.1371/journal.ppat.1006187

Figure Lengend Snippet: (A) Cartoon rendering of how UBXN1 may be interfering with Cul1 in the canonical NFκB signaling pathway; (B) and (C) additional schematics of how UBXN1 may somehow be interfering with Cul1 activity either by steric hindrance (B) or allosteric effect (C) . UBXN1 is known to multimerize and interacts with both N and C termini of Cul1; stoichiometry between UBXN1 and Cul1 is not known (dashed ovals). In both models Skp1 and Rbx1 interact with the N and C termini of Cul1, respectively, and UBXN1 interacts with Rbx1 but not Skp1. These models do not exclude the possibility that another factor or protein (e.g., multimerized ubiquitin) mediates the interaction between UBXN1 and Cul1/Rbx1.

Article Snippet: Rabbit anti-Cul1 monoclonal antibody was from Novus Biologicals (NBP1-40523).

Techniques: Activity Assay, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Characterization of SCF-Complex during Bovine Preimplantation Development

doi: 10.1371/journal.pone.0147096

Figure Lengend Snippet: Primer details.

Article Snippet: Then they were incubated with rabbit anti-cullin 1 antibody (Abgent AJ 1205a, San Diego, CA) 1:100 together with mouse anti-SKP 1 antibody (Abcam 4E11, Cambridge, UK) 1:100, overnight at 4°C.

Techniques: Amplification, Luciferase

Journal: PLoS ONE

Article Title: Characterization of SCF-Complex during Bovine Preimplantation Development

doi: 10.1371/journal.pone.0147096

Figure Lengend Snippet: Untreated embryos. The data were normalised according to the relative concentration of the external standard (luciferase mRNA, 1 pg per oocyte/embryo). (A) Cul 1-like , (B) Cul1 , (C) Skp1 , (D) Rbx1 . Bars show ± S.D. a,b,c,d . Values with different superscripts indicate statistical significance (P<0.05). (MII, MII stage oocyte; 2c, two-cell stage embryo; 4c, four-cell stage embryo; E8c, early eight-cell embryo; L8c, late eight-cell stage; mor, morula; bl, blastocyst).

Article Snippet: Then they were incubated with rabbit anti-cullin 1 antibody (Abgent AJ 1205a, San Diego, CA) 1:100 together with mouse anti-SKP 1 antibody (Abcam 4E11, Cambridge, UK) 1:100, overnight at 4°C.

Techniques: Concentration Assay, Luciferase

Journal: PLoS ONE

Article Title: Characterization of SCF-Complex during Bovine Preimplantation Development

doi: 10.1371/journal.pone.0147096

Figure Lengend Snippet: The data were normalised according to the relative concentration of the external standard (luciferase mRNA, 1pg per embryo). (A) Cul1 –embryos treated from one-cell to two-cell stage, (B) Cul1 –embryos treated from one-cell to four-cell stage, (C) Skp1 – embryos treated from one-cell to four-cell stage, (D) Skp1 –embryos treated from four-cell to early eight-cell stage, (E) Rbx1 –embryos treated from four-cell to early eight-cell stage, (F) Rbx1 –embryos treated from four-cell to late eight-cell stage. Bars show ± S.D. a,b Values with different superscripts indicate statistical significance (P<0.05). (C, control group of untreated embryos; AA, group of embryos treated with α-amanitin; 2c, two-cell stage embryo; 4c, four-cell stage embryo; E8c, early eight-stage embryo; L8c, late eight-cell stage embryo).

Article Snippet: Then they were incubated with rabbit anti-cullin 1 antibody (Abgent AJ 1205a, San Diego, CA) 1:100 together with mouse anti-SKP 1 antibody (Abcam 4E11, Cambridge, UK) 1:100, overnight at 4°C.

Techniques: Concentration Assay, Luciferase

Journal: PLoS ONE

Article Title: Characterization of SCF-Complex during Bovine Preimplantation Development

doi: 10.1371/journal.pone.0147096

Figure Lengend Snippet: 30 embryos per lane. A) Cullin 1; B) SKP1 (antibody 4E11); C) RBX1. The data were processed using Quantity One software (Bio-Rad). 100% represents the sum of the trace quantities of all bands; relative abundance (y-axis) represents the percentage of each band. Bars show mean ± S.D. a,b Values with different superscripts indicate statistical significance (P<0.05). The experiments were repeated at least three times, and representative western blot images are shown below the graph. B’) Representative image of additional bands (approximate size 55 and 90 kDa). (MII, MII oocytes; L8c, late eight-cell stage embryos; 4c, four-cell stage embryos).

Article Snippet: Then they were incubated with rabbit anti-cullin 1 antibody (Abgent AJ 1205a, San Diego, CA) 1:100 together with mouse anti-SKP 1 antibody (Abcam 4E11, Cambridge, UK) 1:100, overnight at 4°C.

Techniques: Software, Western Blot

Journal: PLoS ONE

Article Title: Characterization of SCF-Complex during Bovine Preimplantation Development

doi: 10.1371/journal.pone.0147096

Figure Lengend Snippet: Nuclei (DAPI)–blue; cullin 1 –green.

Article Snippet: Then they were incubated with rabbit anti-cullin 1 antibody (Abgent AJ 1205a, San Diego, CA) 1:100 together with mouse anti-SKP 1 antibody (Abcam 4E11, Cambridge, UK) 1:100, overnight at 4°C.

Techniques:

Journal: PLoS ONE

Article Title: Characterization of SCF-Complex during Bovine Preimplantation Development

doi: 10.1371/journal.pone.0147096

Figure Lengend Snippet: PLA signal indicates Cul1-Skp1 interaction (A’-E’) and the nuclei were stained with DAPI (A-E). In merges (A”-E”), PLA signal is red and DNA blue.

Article Snippet: Then they were incubated with rabbit anti-cullin 1 antibody (Abgent AJ 1205a, San Diego, CA) 1:100 together with mouse anti-SKP 1 antibody (Abcam 4E11, Cambridge, UK) 1:100, overnight at 4°C.

Techniques: Staining