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: Journal of cell science

Article Title: BIM(EL), an intrinsically disordered protein, is degraded by 20S proteasomes in the absence of poly-ubiquitylation.

doi: 10.1242/jcs.058438

Figure Lengend Snippet: Fig. 6. Inhibition of cullin-based E3 ligases does not disrupt turnover of endogenous BIMEL. (A)Cycling HEK293 cells were left untransfected (C) or transfected with empty vector (EV) or FLAG–dnCul1. After 24 hours, whole cell extracts were prepared, fractionated by SDS-PAGE and immunoblotted with antibodies to FLAG (dnCul1), BIM, Cdc25A, cyclin E (cycE) and p27KIP1. (B)Cycling HEK293 cells were transfected with empty vector (EV), HIS–dnUbc12 or FLAG–dnCul1. After 24 hours, whole cell extracts were prepared, fractionated by SDS-PAGE and immunoblotted with antibodies to FLAG (dnCul1), HIS (dnUbc12) or p27KIP1. (C)HR1 cells (HEK293+RAF- 1:ER*) were transfected with empty vector (EV), HIS–dnUbc12 or FLAG– dnCul1. After 24 hours, cells were stimulated with 4-HT + 10M emetine for 6 hours to activate the ERK1/2 pathway. Cell extracts were prepared, fractionated by SDS-PAGE and immunoblotted with antibodies to FLAG (dnCul1), HIS (dnUbc12) or BIM or P-ERK1/2. (D)HR1 cells were left untreated or transfected with control siRNA (siCon) or Cul1-specific siRNA (siCul1). Cells were then treated with 4-HT + 10M emetine for 8 hours or left untreated (C). Cell extracts were fractionated by SDS-PAGE and immunoblotted with antibodies to Cul1, BIM and P-ERK1/2. ERK1 served as a loading control in all experiments.

Article Snippet: For Cul1 knockdown, a pool of three siRNAs targeting Cul1 was used (Santa Cruz Biotechnology) alongside a human siRNA control (Santa Cruz Biotechnology).

Techniques: Inhibition, Transfection, Plasmid Preparation, SDS Page, 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:

Article Title: Structural Insights into NEDD8 Activation of Cullin-RING Ligases: Conformational Control of Conjugation

doi: 10.1016/j.cell.2008.07.022

Figure Lengend Snippet: A, Cul5ctd(green) -Rbx1 (navy) superposition with Cul1-Rbx1 and Cul4A-Rbx1 ctds (1LDJ, olive; 1LDK, orange; ,1U6G, brown; 2HYE, pink) (Angers et al., 2006; Goldenberg et al., 2004; Zheng et al., 2002b). B, Cartoon of Cul5ctd (green)-Rbx1 (navy), indicating positions of Helices 24 and 29 (H24, H29) and the 4HB-, α/β-, and WHB- subdomains. C, Cartoons of the two NEDD8 (yellow, gold)~Cul5ctd (lime, sky)-Rbx1 (blue, violet) complexes in the asymmetric unit, with the Cul5 Lys724~NEDD8 isopeptide bond in sticks. D, Superposition of the 4HB-, α/β-, and WHB-subdomains for the two NEDD8~Cul5ctd-Rbx1 complexes from the au, shown in three orientations, colored as in C. E, Superposition the 4HB-, α/β-subdomains from NEDD8~Cul5ctd-Rbx1s and Cul5ctd-Rbx1, oriented and colored as in B and C.

Article Snippet: Antibodies used for westerns were against Cul1 (Rockland, 100-401-A01; Santa Cruz sc-12761), Rbx1 (Rockland 100-401-A13), His-tag (Qiagen, 34660), p27 (Santa Cruz, sc-527,sc-16324), and biotin (Rockland, 100-4198).

Techniques:

Journal:

Article Title: Structural Insights into NEDD8 Activation of Cullin-RING Ligases: Conformational Control of Conjugation

doi: 10.1016/j.cell.2008.07.022

Figure Lengend Snippet: A, CAND1 (red)-Cul1 (ntd grey; 4HB, sky; α/β, lime; WHB, green)-Rbx1 (RING, blue) structure (Goldenberg et al., 2004), shown as a cartoon and semi-transparent surface, with the 4HB- and α/β-subdomains oriented as in the right view of 1D. B, Cul1-Rbx1 (Goldenberg et al., 2004; Zheng et al., 2002b), colored and oriented as in A, highlighting the CAND1-interacting surface at the junction of the 4HB-, WHB-, and Rbx1 RING subdomains. C, D, Models of full-length NEDD8~CRL structures generated by superimposing the α/β-and ntd-binding 4HB subdomains from NEDD8~Cul5ctd-Rbx1 and Cul1-Rbx1, oriented as in A and B. NEDD8 is yellow, cullin is colored as in A and B, and the two crystallographically-observed Rbx1 conformations for a NEDD8-ligated complex are colored as in 1C. NEDD8 mediated subdomain rearrangement eliminates the indicated CAND1 binding surface (red circle), and positions NEDD8 and its associated WHB to contact the cullin ntd (grey).

Article Snippet: Antibodies used for westerns were against Cul1 (Rockland, 100-401-A01; Santa Cruz sc-12761), Rbx1 (Rockland 100-401-A13), His-tag (Qiagen, 34660), p27 (Santa Cruz, sc-527,sc-16324), and biotin (Rockland, 100-4198).

Techniques: Generated, Binding Assay

Journal:

Article Title: Structural Insights into NEDD8 Activation of Cullin-RING Ligases: Conformational Control of Conjugation

doi: 10.1016/j.cell.2008.07.022

Figure Lengend Snippet: A, Cys pairs (A and B) at Cul1 and Rbx1 residues within 10 Å in the closed conformation, but widely separated in open conformations based on NEDD8~Cul5ctd-Rbx1 crystal structures. B, Western blots probed with anti-Cul1 and anti-Rbx1 after disulfide crosslinking of Cul1 and Rbx1 for unNEDDylated (-NEDD8) and NEDD8ylated (NEDD8) complexes harboring either wild-type or the indicated (A or B) Cys mutants of Cul1 and/or Rbx1. Cys As were in “split ‘n coexpress” Cul1-Rbx1, in which Cul1’s ntd and ctd are copurified as two polypeptides (Zheng et al., 2002b). Cul1 CysB masks the anti-Cul1 C-terminus epitope, and thus CysBs were tested in single polypeptide Cul1 and Rbx1 from insect cells for immunodetection. C, Endoproteinase Glu-C cleavage of a glutamate-rich patch (red) at the Cul1 hinge region in NEDD8ylated (+N8) and unNEDD8ylated (−N8) full-length “split ‘n coexpress” Cul1-Rbx1, with reaction producted detected by western blotting against Cul1 C-terminus (left), NEDD8 (middle), or Cul1 ntd (right).

Article Snippet: Antibodies used for westerns were against Cul1 (Rockland, 100-401-A01; Santa Cruz sc-12761), Rbx1 (Rockland 100-401-A13), His-tag (Qiagen, 34660), p27 (Santa Cruz, sc-527,sc-16324), and biotin (Rockland, 100-4198).

Techniques: Western Blot, Immunodetection

Journal:

Article Title: Structural Insights into NEDD8 Activation of Cullin-RING Ligases: Conformational Control of Conjugation

doi: 10.1016/j.cell.2008.07.022

Figure Lengend Snippet: A, Time-courses of polyubiquitination by SCFs reconstituted with fully-NEDD8ylated wild-type Cul1-Rbx1 (WT~N8), the non-NEDD8ylatable control (K720R), and the Cul1-Rbx1 WHB deletion mutant (ΔWHB). Left - SCFβTRCP-mediated polyubiquitination of a biotin-labeled β-catenin phosphopeptide, detected by western blotting with anti-biotin antisera. Right - SCFSkp2/CksHs1-mediated polyubiquitination of phospho-p27, detected by western blotting with anti-p27 antisera. B, NEDD8ylation time-course for wild-type (wt) Cul1-Rbx1, non-NEDD8ylatable (K720R) control, and variants harboring the indicated Rbx1 linker Val insertion/deletion or Pro mutations, detected by western blotting with anti-Cul1 C-terminus antisera. C, SCFβTRCP-mediated polyubiquitination of a biotin-labeled β-catenin phosphopeptide or D, SCFSkp2/CksHs1-mediated polyubiquitination of phospho-p27 for fully NEDD8ylated SCFs harboring the indicated Rbx1 linker mutants. Pulse-chase Ub(K>R) transfer from UbcH5b for E, SCFβTRCP and a biotin-labeled β-catenin phosphopeptide and F, SCFSkp2/CksHs1 and phospho-p27. G, NEDD8ylation for Rbx1 linker triple Val or Gly insertions (left), and indicated Pro mutants or those with upstream Gly insertions (^ G) (right). H, SCFβTRCP-mediated polyubiquitination of a biotin-labeled β-catenin phosphopeptide and I, SCFSkp2/CksHs1-mediated polyubiquitination of phospho-p27 for fully NEDD8ylated SCFs harboring Rbx1 linker triple Val or Gly insertions (left), and indicated Pro mutants or those with upstream Gly insertions (^ G) (right).

Article Snippet: Antibodies used for westerns were against Cul1 (Rockland, 100-401-A01; Santa Cruz sc-12761), Rbx1 (Rockland 100-401-A13), His-tag (Qiagen, 34660), p27 (Santa Cruz, sc-527,sc-16324), and biotin (Rockland, 100-4198).

Techniques: Control, Mutagenesis, Labeling, Phospho-proteomics, Western Blot, Pulse Chase

Journal:

Article Title: Structural Insights into NEDD8 Activation of Cullin-RING Ligases: Conformational Control of Conjugation

doi: 10.1016/j.cell.2008.07.022

Figure Lengend Snippet: Time-courses of polyubiquitination by A, SCFβTRCP and B, SCFSkp2/CksHs1 reconstituted with fully-NEDD8ylated wild-type Cul1-Rbx1 (WT~N8) or Cul1/5 chimeras (A or B~N8), and their non-NEDD8ylatable counterparts (K>R), as in A. C, NEDD8ylation time-course and CAND1 inhibition for wild type Cul1-Rbx1 and the Cul1/5-Rbx1 chimeras. Reactions were separated by SDS-PAGE and visualized by Coomassie staining.

Article Snippet: Antibodies used for westerns were against Cul1 (Rockland, 100-401-A01; Santa Cruz sc-12761), Rbx1 (Rockland 100-401-A13), His-tag (Qiagen, 34660), p27 (Santa Cruz, sc-527,sc-16324), and biotin (Rockland, 100-4198).

Techniques: Inhibition, SDS Page, Staining

Journal:

Article Title: Structural Insights into NEDD8 Activation of Cullin-RING Ligases: Conformational Control of Conjugation

doi: 10.1016/j.cell.2008.07.022

Figure Lengend Snippet: A, Polyubiquitination reactions with SCFβTRCP/β-catenin phosphopeptide (left), and SCFSkp2/CksHs1/ phospho-p27 (right) reconstituted with non-NEDD8ylatable (K720R), un-NEDD8ylated wild-type (WT), and fully NEDD8ylated Cul1-Rbx1 (WT~N8). Reaction products were detected by immunoblotting, top panels with anti-biotin (left) or anti-p27 (right), middle with anti-His (Ubiquitin; green * - Cul1~Ubiquitin), and lower with anti-Cul1 C-terminus antisera. B–E, Models of conformations involved in CRL activities. B, Structural model of SCFβTRCP-β-catenin phosphopeptide complex (Wu et al., 2003; Zheng et al., 2002b) with Rbx1 RING and E2 docked based on the c-Cbl-UbcH7 structure (Zheng et al., 2000), with the generic E2 in light blue with catalytic Cys a yellow sphere, Rbx1 in blue, the ctd portion of Cul1 in green, Cul1 ntd in grey, the substrate adaptor Skp1- F-box protein (Fbp) βTRCP complex in purple and magenta, and β–catenin peptide target in green sticks. Approximate distances to be spanned for NEDD8 transfer to the cullin target Lys (green stick) and for NEDD8 transfer to target during polyubiquitination are indicated with arrows. C, Model of cullin ligation to NEDD8 (yellow), where the NEDD8 E2 (cyan) Cys and cullin target lysine are justaposed via rotation of the Rbx1 linker (residues 36–41). D, Model of ubiquitin (orange) transfer from ubiquitin E2 (teal) to target by a NEDD8ylated CRL, generated by superimposing the α/β-and ntd-binding 4HB subdomains from NEDD8~Cul5ctd-Rbx1 and Cul1-Rbx1. The E2 Cys and peptide target brought into proximity via rotation of the Rbx1 linker. E, Model of target polyubiquitination by a NEDD8ylated CRL, where rotation about the Rbx1 linker allows multiple catalytic geometries associated polyubiquitin chain extension.

Article Snippet: Antibodies used for westerns were against Cul1 (Rockland, 100-401-A01; Santa Cruz sc-12761), Rbx1 (Rockland 100-401-A13), His-tag (Qiagen, 34660), p27 (Santa Cruz, sc-527,sc-16324), and biotin (Rockland, 100-4198).

Techniques: Phospho-proteomics, Western Blot, Ubiquitin Proteomics, Ligation, Generated, Binding Assay