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: The Journal of Clinical Investigation

Article Title: A haploinsufficiency restoration strategy corrects neurobehavioral deficits in Nf1 +/– mice

doi: 10.1172/JCI188932

Figure Lengend Snippet: ( A and B ) HeLa cells transfected with an empty expression vector or with vectors containing either an FBXW11 cDNA or an FBXO3 cDNA were collected and lysed 48 hours later. NF1, FBXW11, and FBXO3 proteins were detected by immunoblotting, with GAPDH used as a loading control. ( C ) Nf1 +/– MEFs were treated with the FBXW11 inhibitor PDTC (50 μM) or the FBXO inhibitor BC-1215 (20 μg/mL) for 6 hours prior to harvesting for immunoblotting. The lower panels represent densitometric analysis comparing NF1 levels with the GAPDH control. * P < 0.05 and ** P < 0.01, by unpaired 2-tailed t test. Data indicate the mean ± SEM.

Article Snippet: Synthesized ANVDSGLMHSIGLGYHK peptides (Selleckchem), MG132 (Tocris), bortezomib (Selleckchem), CHX (MilliporeSigma), BC-1215 (MilliporeSigma), pyrrolidinedithiocarbamate ammonium (PDTC) (P8765, MilliporeSigma), siPORT NeoFX Transfection Reagent (Ambion), ECL Western blotting detection reagent (Amersham Biosciences), IPP: 200 units/mg (catalog 10108987001, Roche), E2: His-UbcH3/Cdc34, human recombinant (catalog E2-610, BostonBiochem); Flag peptide (3290, MilliporeSigma), and polyethylenimine (PEI) 25 kDa linear (catalog 23966-2, Polysciences); human F-box siRNA library (Ambion), human FBXW11 siRNA (siRNA ID 23487, Ambion), human FBXO3 siRNA (siRNA ID 25346, Ambion), human FBXW11-3 unique 27 mer siRNA duplexes (SR308161, Origene), human FBXW1/b-TrCP1 siRNA (SASI_Hs01_00189438 and SASI_Hs01_00189439, MilliporeSigma), and Mission siRNA universal negative control 1 (SIC002, MilliporeSigma); GFP-tagged human neurofibromin, transcript variant 1 cDNA (RG220425, Origene), Myc-DDK–tagged human FBXO3 transcript variant 1 cDNA (RC208494, Origene), Myc-DDK–tagged human FBXW11 transcript variant 3 cDNA (RC218905, Origene), Myc-DDK–tagged human FBXW7 transcript variant 1 cDNA (RC217398, Origene), human RBX1 cDNA (SC115112, Origene), human SKP1 transcript variant 1 cDNA (SC126980, Origene), and human CUL1 cDNA (SC108409, Origene); human FBXW11 recombinant protein (H00023291-P01, Abnova), human CUL1 recombinant protein (H00008454-P01, Abnova), human RBX1 recombinant protein (H00009978-P01, Abnova), and human SKP1 recombinant protein (H00006500-P01, Abnova).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Control

Journal: The Journal of Clinical Investigation

Article Title: A haploinsufficiency restoration strategy corrects neurobehavioral deficits in Nf1 +/– mice

doi: 10.1172/JCI188932

Figure Lengend Snippet: ( A ) Schematic map of the neurofibromin (NF1) interaction domains used to test interactions with FBXW11. Full-length NF1 was divided into the 6 indicated domains and subcloned into GFP-expressing plasmids. Flag-tagged FBXW11 and the various GFP-tagged NF1 subdomains were transfected into HEK293T cells. Six hours prior to harvesting, the cells were treated with 15 μM MG-132. Anti-Flag IP followed by immunoblotting was used to detect GFP-tagged NF1 co-IP with FBXW11. Relative binding activity as determined by immunoblotting is indicated. Image created in BioRender. Angus, S. (2025) https://BioRender.com/n28p603 ( B ) HEK293T cells were treated as in A to confirm the specific interaction of FBXW11 with the domain 3 (D3) fragment of the NF1 peptide, which encompasses the GRD of NF1 isoform 1 (GRD1), but not GRD2. ( C ) A NanoBiT complementation assay was performed after cotransfection of HEK293T cells with LgBiT-FBXW11 and SmBiT-GRD1 or GRD2 fusion proteins. Forty-five hours after transfection, the interaction was detected by luminescence. The negative control (neg. cont.) refers to luminescence values obtained from wells containing cells with LgbiT-FBXW11 and SmBiT-empty. Vehicle or the proteasome inhibitor bortezomib (1 mM) was included to enhance the interaction due to GRD1 accumulation. * P < 0.05 and *** P < 0.001, by unpaired 2-tailed t test. Data indicate the mean ± SEM. Image created in BioRender. Angus, S. (2025) https://BioRender.com/e61l197 ( D ) The SCF complex (SKP1, CUL1, RBX1, and Flag-FBXW11) was purified after cotransfection and subsequent Flag-IP (and Flag-peptide elution) from HEK293T cell lysates. Isolated GFP-GRD1 was incubated at 37°C for 60 minutes in the presence of E1 (100 nM), E2 (2 mM), Mg-ATP (5 mM), and ubiquitin (Ub) in the presence (+) or absence (–) of SCF FBXW11 . Samples were then subjected to immunoblotting, and GRD1 ubiquitination was detected by GFP antibody and the smearing due to higher-molecular-weight species. ( E ) HEK293T cells were transfected with control or FBXW11 -targeting siRNAs for 72 hours. A TUBEs assay was performed using agarose-TUBE2 to pull down total ubiquitinated protein. Immunoblotting was used to detect ubiquitin and NF1.

Article Snippet: Synthesized ANVDSGLMHSIGLGYHK peptides (Selleckchem), MG132 (Tocris), bortezomib (Selleckchem), CHX (MilliporeSigma), BC-1215 (MilliporeSigma), pyrrolidinedithiocarbamate ammonium (PDTC) (P8765, MilliporeSigma), siPORT NeoFX Transfection Reagent (Ambion), ECL Western blotting detection reagent (Amersham Biosciences), IPP: 200 units/mg (catalog 10108987001, Roche), E2: His-UbcH3/Cdc34, human recombinant (catalog E2-610, BostonBiochem); Flag peptide (3290, MilliporeSigma), and polyethylenimine (PEI) 25 kDa linear (catalog 23966-2, Polysciences); human F-box siRNA library (Ambion), human FBXW11 siRNA (siRNA ID 23487, Ambion), human FBXO3 siRNA (siRNA ID 25346, Ambion), human FBXW11-3 unique 27 mer siRNA duplexes (SR308161, Origene), human FBXW1/b-TrCP1 siRNA (SASI_Hs01_00189438 and SASI_Hs01_00189439, MilliporeSigma), and Mission siRNA universal negative control 1 (SIC002, MilliporeSigma); GFP-tagged human neurofibromin, transcript variant 1 cDNA (RG220425, Origene), Myc-DDK–tagged human FBXO3 transcript variant 1 cDNA (RC208494, Origene), Myc-DDK–tagged human FBXW11 transcript variant 3 cDNA (RC218905, Origene), Myc-DDK–tagged human FBXW7 transcript variant 1 cDNA (RC217398, Origene), human RBX1 cDNA (SC115112, Origene), human SKP1 transcript variant 1 cDNA (SC126980, Origene), and human CUL1 cDNA (SC108409, Origene); human FBXW11 recombinant protein (H00023291-P01, Abnova), human CUL1 recombinant protein (H00008454-P01, Abnova), human RBX1 recombinant protein (H00009978-P01, Abnova), and human SKP1 recombinant protein (H00006500-P01, Abnova).

Techniques: Expressing, Transfection, Western Blot, Co-Immunoprecipitation Assay, Binding Assay, Activity Assay, Cotransfection, Negative Control, Purification, Isolation, Incubation, Ubiquitin Proteomics, Molecular Weight, Control

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: 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