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

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: PLoS ONE

Article Title: Myeloma Overexpressed 2 (Myeov2) Regulates L11 Subnuclear Localization through Nedd8 Modification

doi: 10.1371/journal.pone.0065285

Figure Lengend Snippet: ( A ) Protein identified by mass spectrometry of Myeov2 immunoprecipitants. The colored squares show how often a protein was identified in multiple experiments. ( B ) Co-immunoprecipitation of HA-Myeov2 and Flag-CSN5 in HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and analyzed using Flag and HA antibodies. ( C ) Co-immunoprecipitation of Flag-Myeov2 and endogenous CSN5 in HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and analyzed using Flag and CSN5 antibodies. ( D ) Schematic structure of Myeov2 deletion mutants. ( E ) Co-immunoprecipitation of CSN5, Cul1, and Cul3 with Flag-Myeov2 in HEK293 cells. Cell lysates were immunoprecipitated with anti-Flag antibody and analyzed using indicated antibodies.

Article Snippet: Antibodies to Flag (M2, SIGMA), Myc (9E10 Santa Cruz), HA (A190-108A, Bethyl Lab., Inc), HA (Y-11, Santa Cruz), CSN5 (42, BD Transduction Lab.), Cul1 (19/CUL-1, BD Transduction Lab.), Tubulin (DM1A, SIGMA), His (GE Healthcare), p53 (DO-1, Santa Cruz), and Mdm2 (N-20, Santa Cruz) were used for immunoblot analyses.

Techniques: Mass Spectrometry, Immunoprecipitation

Journal: Experimental & Molecular Medicine

Article Title: Upregulation of miR-223 in the rat liver inhibits proliferation of hepatocytes under simulated microgravity

doi: 10.1038/emm.2017.80

Figure Lengend Snippet: The effect of SMG on hepatocyte proliferation. ( a ) Immunostaining analysis of Ki67 (left panels), PCNA (center panels) and PH3 (right panels) in the rat liver of the control, TS-14, TS-28 and TS-42 groups. Scale bar, 50 μm. ( b ) Quantification of the Ki67-, PCNA- and PH3-positive cell number in the liver of the control, TS-14, TS-28 and TS-42 groups, n =6. ( c ) RT-qPCR analysis of gene expression related to cell cycle regulation such as CCNA2 , CCND1 , CDK1 , CDK2 and FOXM1 in the rat liver of the control and TS groups. n =6. ( d ) Protein levels of PCNA, cyclin D1, cyclin D3 and CDK2 were analyzed by western blotting. n =3. The intensity of the protein bands was semi-quantitatively analyzed using ImageJ software. * P <0.05, ** P <0.01 compared to Control. SMG, simulated microgravity; TS, tail suspension.

Article Snippet: Specific antibodies to p27, CDK2, CUL1 (ABclonal Technology), PCNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cyclin D1 (Invitrogen), cyclin D3 and GAPDH (Cell Signaling Technology) were used.

Techniques: Immunostaining, Control, Quantitative RT-PCR, Gene Expression, Western Blot, Software, Suspension

Journal: Experimental & Molecular Medicine

Article Title: Upregulation of miR-223 in the rat liver inhibits proliferation of hepatocytes under simulated microgravity

doi: 10.1038/emm.2017.80

Figure Lengend Snippet: CDK2 and CUL1 were the direct targets of miR-223. Schematic representation of the luciferase constructs used for reporter assays. The miR-223 target sites within the 3′-UTR of CDK2 ( a ) and CUL1 ( b ) are shown as black boxes. The sequences below indicated the putative miR-223 target sites on the wild-type (WT) and mutated (MUT) constructs. The miR-223-binding sites and seed sequences are marked with bold italics. ( c , d ) Effects of miR-223 on the luciferase activity of the reporter gene in the WT or mutated vector in Hepa1-6 cells. n =3. ( e ) CDK2 and CUL1 protein expression in Hepa1-6 cells was examined after transfection with miR-233 mimic and negative control for 48 h and semi-quantitative evaluation using ImageJ software. mRNA ( f ) and protein ( g ) expression of CUL1 analyzed in the rat liver of the control and TS groups. n =6, * P <0.05, ** P <0.01 compared to Control.

Article Snippet: Specific antibodies to p27, CDK2, CUL1 (ABclonal Technology), PCNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cyclin D1 (Invitrogen), cyclin D3 and GAPDH (Cell Signaling Technology) were used.

Techniques: Luciferase, Construct, Binding Assay, Activity Assay, Plasmid Preparation, Expressing, Transfection, Negative Control, Software, Control

Journal: Experimental & Molecular Medicine

Article Title: Upregulation of miR-223 in the rat liver inhibits proliferation of hepatocytes under simulated microgravity

doi: 10.1038/emm.2017.80

Figure Lengend Snippet: Upregulation of p27 expression in vitro and in vivo . ( a ) Immunofluorescence staining of p27 in the rat liver of the control, TS-14, TS-28 and TS-42 groups. Images were captured by a Leica TCS SP5 confocal scanning laser microscope. p27 in red (Cy3); nuclei in blue (DAPI). Scale bar, 50 μm. ( b ) Quantification of the number of p27-positive cells in the liver of the control, TS-14, TS-28 and TS-42 groups. n =6. ( c ) p27 protein expression level in the TS liver was significantly elevated compared with that of the control as shown by western blotting and semi-quantitative evaluation using ImageJ software. ( d ) The expression of the p27 protein in Hepa1-6 cells was examined 48 h after transfection with miR-233 mimic and negative control by western blotting and semi-quantitatively evaluated using ImageJ software. * P <0.05, ** P <0.01 compared to Control. ( e ) Diagram illustrating the regulatory hierarchy among miR-223 and its targets CDK2 and CUL1 in the control of hepatocyte proliferation.

Article Snippet: Specific antibodies to p27, CDK2, CUL1 (ABclonal Technology), PCNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cyclin D1 (Invitrogen), cyclin D3 and GAPDH (Cell Signaling Technology) were used.

Techniques: Expressing, In Vitro, In Vivo, Immunofluorescence, Staining, Control, Microscopy, Western Blot, Software, Transfection, Negative Control

Journal: Biomolecules

Article Title: CSN5A Subunit of COP9 Signalosome Temporally Buffers Response to Heat in Arabidopsis

doi: 10.3390/biom9120805

Figure Lengend Snippet: Increased growth of csn5a-1 following 7 d heat treatment is due to an increase in deneddylation activity (against CUL1) and auxin response. ( a ) Immunoblot using equal protein concentrations with CSN5 antibodies did not show any change in the protein band of csn5a-1 after heat treatment. ( b ) Immunoblot using equal protein concentrations with CUL3 antibodies did not show any change in cullin neddylation/deneddylation (93/85 kDa) ratio in either in csn5a-1 or Col-0 after heat treatment. ( c ) Immunoblot using equal protein concentrations with CUL1 showed increase in deneddylation activity of csn5a-1 and Col-0. Rubisco large subunit stained with ponceau is used as loading control. NT, non-treated. ( d ) Expression of auxin responsive genes (SAUR19 (Small auxin-up RNA 19) and EXPA4 (Expansin A4) of which the expression was downregulated in csn5a-1 control plants compared to WT (Col-0) control plants increases after heat treatment. Expression of WT (Col-0) control plant is taken as the baseline (0) in the log2 fold change. Error bars represent SEM of biological replicates ( n = 3–5). Student’s t test * p < 0.05; ** p < 0.01.

Article Snippet: Protein concentration was determined by Bradford protein assay, and equal amounts of protein were loaded for western blot with specific antibodies anti-CSN5 (Abcam, ab195635, Cambridge, UK), anti-CUL3 (Enzo, BML-PW0470, Enzo Life Sciences, NY, USA), and anti-CUL1 (Enzo, BML-PW0190).

Techniques: Activity Assay, Western Blot, Staining, Expressing

Journal: Leukemia

Article Title: A genome-scale CRISPR-Cas9 screening in myeloma cells identifies regulators of immunomodulatory drug sensitivity

doi: 10.1038/s41375-018-0205-y

Figure Lengend Snippet: Inactivation of SCFFbxo7 restores CRBN levels and IMiDs sensitivity of CSN knockout MM cells. a Analysis of the levels of neddylated Cul1 and Cul4A in CSN2 deleted MM.1S cells. The asterisk indicates a non-specific band. b, c Immunoblot analysis of CRBN levels in MM.1S cells with CSN2/6 single knockout or CSN2/6-Cul1 (b) or CSN2/6-Fbxo7 (c) double knockout. The asterisk indicates a non-specific band. d, e Dose-dependent survival of MM cells with Cul1 single knockout or CSN2/6-Cul1 double knockout (d) and MM cells with Fbxo7 single knockout or CSN2/6-Fbxo7 double knockout (e) treated with Poma. Error bars represent s.e.m. (n = 3)

Article Snippet: Anti-Flag (F3165; Sigma-Aldrich), anti-HA (H6908; H3663; Sigma- Aldrich), anti-Myc (2272; Cell Signaling Technology), anti- Cul1 (AP16324b; Abgent), anti-β-actin (5779–1; Epi- tomics), anti-actin (1844–1; Epitomics), anti-TRIP15 (ab155774; Abcam), anti-CSN6 (NBP1–79752), anti-JAB1 (ab182756; Abcam), anti-CSN7B (ab133548; Abcam), anti- CSN7A (AP12810b; Abgent), anti-CSN8 (AP14987a; Abgent), anti-Fbxo7 (ab57037; Abcam), anti-IKZF3 (ab139408; Abcam), anti-IKZF1 (sc-13039; Santa Cruz), anti-Nedd8 (ab81264; Abcam), anti-HA agarose (E6779; Sigma-Aldrich), anti-Myc agarose (E6654; Sigma-Aldrich), and anti-CRBN (SAB045910; Sigma-Aldrich) were purchased and used according to the manufacturers’ recommendations.

Techniques: Knock-Out, Western Blot, Double Knockout