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: Antibodies used included: anti-MsrA antibody (Proteintech Group, Rosemont, IL, USA), anti-Jab1 antibody (Thermo-Fisher Scientific, Waltham, MA, USA), anti-Cul-1 antibody (Novus, Littleton, CO, USA), anti-Nedd8 antibody (Boster Bio, Pleasanton, CA, USA), anti-P27 antibody (Proteintech Group, Rosemont, IL, USA), anti-β actin antibody (Abcam, Cambridge, UK), and HRP-conjugated secondary antibodies for Western blot analyses (Rabbit anti-mouse and Goat anti-rabbit) (Bio-Rad, Hercules, CA, USA).

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

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: 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: Viruses

Article Title: βTrCP is Required for HIV-1 Vpu Modulation of CD4, GaLV Env, and BST-2/Tetherin

doi: 10.3390/v10100573

Figure Lengend Snippet: Vpu antagonism of GaLV Env is Cullin1 dependent. ( A ) 293FT cells were transfected HIV-CMV-GFP along with either MLV Env or MLV/GaLV Env Δ8. 24 h post-transfection cells were treated with 1 μM MLN4924, or DMSO. Media was collected after 24 h and was transferred to 293T mCAT-1 cells and infectivity was measured by flow cytometry 48 h later. Infectious particle output was normalized to that of HIV-CMV-GFP (Vpu − )/DMSO. ( B ) 293FT cells were transfected with HIV-CMV-GFP along with either MLV Env or MLV/GaLV Env Δ8 and either DN Cullin1, DN βTrCP, or empty vector control. After 48 h, media was transferred to 293T mCAT-1 cells and infectivity was measured by flow cytometry 48 h later. ( C ) 293FT cells were transfected with human CD4 alone, CD4 + Vpu IRES GFP, or CD4 + Vpu IRES GFP + DN Cullin1 or DN βTrCP. CD4 surface protein was labeled and analyzed by flow cytometry 48 h later. Asterisks indicate p < 0.01.

Article Snippet: The pHAGE DN Cullin1 was a gift from Stephen Elledge (Addgene plasmid # 41911) [ ].

Techniques: Transfection, Infection, Flow Cytometry, Plasmid Preparation, Control, Labeling