cul3nδ22 Search Results


human cul3nδ22  (Addgene inc)
90
Addgene inc human cul3nδ22
Figure 1. A55 directly binds to cullin-3 via its N-terminal BB domain. A-C. Representative immunoblots following immunoprecipitation (IP) of cleared lysates from HEK293T-REx cell lines (A) expressing empty vector (EV), B14-TAP or TAP-A55, the TAP tag comprising STREP and FLAG epitopes; (B) expressing B14-TAP or TAP-A55 and transfected with plasmids encoding myc-Cul3 or myc-Cul5; (C) expressing EV, B14-TAP, TAP-A55, TAP-A55BB or TAP-A55 Kelch. Cells were lysed in (A and C) NP40 or (B) RIPA buffer. Immunoprecipitates were subjected to SDS-PAGE and immunoblotting. A and C. FLAG IP and immunoblotting for co-IP of endogenous Cul3. B. Myc IP and immunoblotting for co-IP of TAP-tagged B14 or A55. Input, cleared lysate. Data shown are representative of at least three independent experiments. Signals arising from the light chain (*) or heavy chain (**) of the antibody used for IP are marked. D. SEC-MALS analyses showing the SEC elution profiles (thin lines) and molecular mass distribution (thick lines) across the elution peaks for A55BB (peak 1, green, theoretical molecular mass 30 kDa, observed molecular mass 60 kDa), <t>Cul3NΔ22</t> (peak 2, blue, theoretical molecular mass 46 kDa, observed molecular mass 45 kDa) and A55BB and Cul3NΔ22 together (peak 3, red, theoretical molecular mass 76 kDa, observed molecular mass 141 kDa) when eluting from a Superdex 200 10/300 GL column. Peak 4 is assumed to be excess Cul3NΔ22. E. Coomassie-stained SDS-PAGE analysis of peaks 1–3 from (D).
Human Cul3nδ22, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pnic-cthf-cul3nδ22  (Addgene inc)
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Addgene inc pnic-cthf-cul3nδ22
Figure 1. A55 directly binds to cullin-3 via its N-terminal BB domain. A-C. Representative immunoblots following immunoprecipitation (IP) of cleared lysates from HEK293T-REx cell lines (A) expressing empty vector (EV), B14-TAP or TAP-A55, the TAP tag comprising STREP and FLAG epitopes; (B) expressing B14-TAP or TAP-A55 and transfected with plasmids encoding myc-Cul3 or myc-Cul5; (C) expressing EV, B14-TAP, TAP-A55, TAP-A55BB or TAP-A55 Kelch. Cells were lysed in (A and C) NP40 or (B) RIPA buffer. Immunoprecipitates were subjected to SDS-PAGE and immunoblotting. A and C. FLAG IP and immunoblotting for co-IP of endogenous Cul3. B. Myc IP and immunoblotting for co-IP of TAP-tagged B14 or A55. Input, cleared lysate. Data shown are representative of at least three independent experiments. Signals arising from the light chain (*) or heavy chain (**) of the antibody used for IP are marked. D. SEC-MALS analyses showing the SEC elution profiles (thin lines) and molecular mass distribution (thick lines) across the elution peaks for A55BB (peak 1, green, theoretical molecular mass 30 kDa, observed molecular mass 60 kDa), <t>Cul3NΔ22</t> (peak 2, blue, theoretical molecular mass 46 kDa, observed molecular mass 45 kDa) and A55BB and Cul3NΔ22 together (peak 3, red, theoretical molecular mass 76 kDa, observed molecular mass 141 kDa) when eluting from a Superdex 200 10/300 GL column. Peak 4 is assumed to be excess Cul3NΔ22. E. Coomassie-stained SDS-PAGE analysis of peaks 1–3 from (D).
Pnic Cthf Cul3nδ22, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Figure 1. A55 directly binds to cullin-3 via its N-terminal BB domain. A-C. Representative immunoblots following immunoprecipitation (IP) of cleared lysates from HEK293T-REx cell lines (A) expressing empty vector (EV), B14-TAP or TAP-A55, the TAP tag comprising STREP and FLAG epitopes; (B) expressing B14-TAP or TAP-A55 and transfected with plasmids encoding myc-Cul3 or myc-Cul5; (C) expressing EV, B14-TAP, TAP-A55, TAP-A55BB or TAP-A55 Kelch. Cells were lysed in (A and C) NP40 or (B) RIPA buffer. Immunoprecipitates were subjected to SDS-PAGE and immunoblotting. A and C. FLAG IP and immunoblotting for co-IP of endogenous Cul3. B. Myc IP and immunoblotting for co-IP of TAP-tagged B14 or A55. Input, cleared lysate. Data shown are representative of at least three independent experiments. Signals arising from the light chain (*) or heavy chain (**) of the antibody used for IP are marked. D. SEC-MALS analyses showing the SEC elution profiles (thin lines) and molecular mass distribution (thick lines) across the elution peaks for A55BB (peak 1, green, theoretical molecular mass 30 kDa, observed molecular mass 60 kDa), Cul3NΔ22 (peak 2, blue, theoretical molecular mass 46 kDa, observed molecular mass 45 kDa) and A55BB and Cul3NΔ22 together (peak 3, red, theoretical molecular mass 76 kDa, observed molecular mass 141 kDa) when eluting from a Superdex 200 10/300 GL column. Peak 4 is assumed to be excess Cul3NΔ22. E. Coomassie-stained SDS-PAGE analysis of peaks 1–3 from (D).

Journal: Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.ra118.006561

Figure Lengend Snippet: Figure 1. A55 directly binds to cullin-3 via its N-terminal BB domain. A-C. Representative immunoblots following immunoprecipitation (IP) of cleared lysates from HEK293T-REx cell lines (A) expressing empty vector (EV), B14-TAP or TAP-A55, the TAP tag comprising STREP and FLAG epitopes; (B) expressing B14-TAP or TAP-A55 and transfected with plasmids encoding myc-Cul3 or myc-Cul5; (C) expressing EV, B14-TAP, TAP-A55, TAP-A55BB or TAP-A55 Kelch. Cells were lysed in (A and C) NP40 or (B) RIPA buffer. Immunoprecipitates were subjected to SDS-PAGE and immunoblotting. A and C. FLAG IP and immunoblotting for co-IP of endogenous Cul3. B. Myc IP and immunoblotting for co-IP of TAP-tagged B14 or A55. Input, cleared lysate. Data shown are representative of at least three independent experiments. Signals arising from the light chain (*) or heavy chain (**) of the antibody used for IP are marked. D. SEC-MALS analyses showing the SEC elution profiles (thin lines) and molecular mass distribution (thick lines) across the elution peaks for A55BB (peak 1, green, theoretical molecular mass 30 kDa, observed molecular mass 60 kDa), Cul3NΔ22 (peak 2, blue, theoretical molecular mass 46 kDa, observed molecular mass 45 kDa) and A55BB and Cul3NΔ22 together (peak 3, red, theoretical molecular mass 76 kDa, observed molecular mass 141 kDa) when eluting from a Superdex 200 10/300 GL column. Peak 4 is assumed to be excess Cul3NΔ22. E. Coomassie-stained SDS-PAGE analysis of peaks 1–3 from (D).

Article Snippet: Human Cul3NΔ22 (Uniprot Q13618, residues 23-388) and Cul3N (Uniprot Q13618, residues 1-388) with the I342R and L346D stabilizing mutations (6) in pNIC-CTHF with C-terminal TEV-cleavable His6-tags were a gift from Nicola BurgessBrown (Addgene plasmids 53672 and 53673).

Techniques: Western Blot, Immunoprecipitation, Expressing, Plasmid Preparation, Transfection, SDS Page, Co-Immunoprecipitation Assay, Staining

Figure 2. ITC studies show that A55 binds to Cul3 with nanomolar to sub-nanomolar affinity. A- D. Representative ITC titration curves showing interactions between A55BB and (A) Cul3NΔ22 or (B) Cul3N and between KLHL3, a human BTB related to A55, and (C) Cul3NΔ22 or (D) Cul3N. The top figure in each panel shows the baseline-corrected differential power (DP) versus time. The bottom figure of each panel is the normalized binding curve showing integrated changes in enthalpy (ΔH) against molar ratio. The corresponding dissociation constant (KD), number of binding sites (N), enthalpy change (ΔH) and change in Gibbs free energy (ΔG) for each representative experiment are shown. All experiments were performed at least twice independently.

Journal: Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.ra118.006561

Figure Lengend Snippet: Figure 2. ITC studies show that A55 binds to Cul3 with nanomolar to sub-nanomolar affinity. A- D. Representative ITC titration curves showing interactions between A55BB and (A) Cul3NΔ22 or (B) Cul3N and between KLHL3, a human BTB related to A55, and (C) Cul3NΔ22 or (D) Cul3N. The top figure in each panel shows the baseline-corrected differential power (DP) versus time. The bottom figure of each panel is the normalized binding curve showing integrated changes in enthalpy (ΔH) against molar ratio. The corresponding dissociation constant (KD), number of binding sites (N), enthalpy change (ΔH) and change in Gibbs free energy (ΔG) for each representative experiment are shown. All experiments were performed at least twice independently.

Article Snippet: Human Cul3NΔ22 (Uniprot Q13618, residues 23-388) and Cul3N (Uniprot Q13618, residues 1-388) with the I342R and L346D stabilizing mutations (6) in pNIC-CTHF with C-terminal TEV-cleavable His6-tags were a gift from Nicola BurgessBrown (Addgene plasmids 53672 and 53673).

Techniques: Titration, Binding Assay

Figure 3. A55 and cellular BB domains share conserved modes of dimerization and Cul3 binding. A. Structure of the A55/Cul3NΔ22 heterodimer in the asymmetric unit as ribbon diagram. Cul3 is in cyan and the three domains of A55 (BTB, 3-box and BACK) are in green, orange and grey, respectively. Helices α1-α12 from A55 are labelled in black with the exception of α10, which is hidden behind α9 in the picture. Helices α1-α16 from Cul3 are labelled in red. B. A55/Cul3 dimer formed by applying crystallographic 2-fold symmetry. C. An overlay of three BB/Cul3 complex structures (KLHL3/Cul3, PDB code 4HXI (7); KLHL11/Cul3, PDB code 4APF (6); A55/Cul3). The structures are aligned to the Cul3 part of the A55/Cul3 complex only. A55, KLHL3, KLHL11 and Cul3 are in green, purple, magenta and cyan, respectively, and the three sub-domains are marked. Additional helices at the C terminus of the KLHL11 BACK domain are shown as semi-transparent helices. D. Comparison of the dimers formed by A55, KLHL3 and KLHL11 BB domains, colored as in (C).

Journal: Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.ra118.006561

Figure Lengend Snippet: Figure 3. A55 and cellular BB domains share conserved modes of dimerization and Cul3 binding. A. Structure of the A55/Cul3NΔ22 heterodimer in the asymmetric unit as ribbon diagram. Cul3 is in cyan and the three domains of A55 (BTB, 3-box and BACK) are in green, orange and grey, respectively. Helices α1-α12 from A55 are labelled in black with the exception of α10, which is hidden behind α9 in the picture. Helices α1-α16 from Cul3 are labelled in red. B. A55/Cul3 dimer formed by applying crystallographic 2-fold symmetry. C. An overlay of three BB/Cul3 complex structures (KLHL3/Cul3, PDB code 4HXI (7); KLHL11/Cul3, PDB code 4APF (6); A55/Cul3). The structures are aligned to the Cul3 part of the A55/Cul3 complex only. A55, KLHL3, KLHL11 and Cul3 are in green, purple, magenta and cyan, respectively, and the three sub-domains are marked. Additional helices at the C terminus of the KLHL11 BACK domain are shown as semi-transparent helices. D. Comparison of the dimers formed by A55, KLHL3 and KLHL11 BB domains, colored as in (C).

Article Snippet: Human Cul3NΔ22 (Uniprot Q13618, residues 23-388) and Cul3N (Uniprot Q13618, residues 1-388) with the I342R and L346D stabilizing mutations (6) in pNIC-CTHF with C-terminal TEV-cleavable His6-tags were a gift from Nicola BurgessBrown (Addgene plasmids 53672 and 53673).

Techniques: Binding Assay, Comparison

Figure 5. Conserved and non-conserved interactions at the interface between A55 and Cul3. A. The A55BB/Cul3NΔ22 complex structure with two key Cul3 binding sites in the BTB domain boxed in black (enlarged in B to F) and red (enlarged in G). B. Structural overlay of the ϕ-X-D/E motifs from A55, KLHL3, KLHL11 and SPOP. C-F. Surface of Cul3 colored by residue hydrophobicity from yellow (hydrophobic) to white (polar) (70). Hydrophobic binding pockets are shown for F54 of A55, M83 of KLHL3, F130 of KLHL11, and M233 of SPOP, which are equivalent to the ϕ residue of the ϕ- X-D/E motif, and for I48 of A55 and its equivalent residues A77, P124 and A227 in KLHL3, KLHL11 and SPOP, respectively. G. An overlay of the hydrogen bond formed between Y125 of Cul3 and D99 of A55 with equivalent residues S128, D181 and D278 in KLHL3, KLHL11 and SPOP, respectively.

Journal: Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.ra118.006561

Figure Lengend Snippet: Figure 5. Conserved and non-conserved interactions at the interface between A55 and Cul3. A. The A55BB/Cul3NΔ22 complex structure with two key Cul3 binding sites in the BTB domain boxed in black (enlarged in B to F) and red (enlarged in G). B. Structural overlay of the ϕ-X-D/E motifs from A55, KLHL3, KLHL11 and SPOP. C-F. Surface of Cul3 colored by residue hydrophobicity from yellow (hydrophobic) to white (polar) (70). Hydrophobic binding pockets are shown for F54 of A55, M83 of KLHL3, F130 of KLHL11, and M233 of SPOP, which are equivalent to the ϕ residue of the ϕ- X-D/E motif, and for I48 of A55 and its equivalent residues A77, P124 and A227 in KLHL3, KLHL11 and SPOP, respectively. G. An overlay of the hydrogen bond formed between Y125 of Cul3 and D99 of A55 with equivalent residues S128, D181 and D278 in KLHL3, KLHL11 and SPOP, respectively.

Article Snippet: Human Cul3NΔ22 (Uniprot Q13618, residues 23-388) and Cul3N (Uniprot Q13618, residues 1-388) with the I342R and L346D stabilizing mutations (6) in pNIC-CTHF with C-terminal TEV-cleavable His6-tags were a gift from Nicola BurgessBrown (Addgene plasmids 53672 and 53673).

Techniques: Binding Assay, Residue

Figure 6. I48E mutation significantly impairs A55 binding to Cul3. A. Representative thermal melt curves of wild-type A55BB and mutants F54E, D99A, I48A and I48E from differential scanning fluorimetry (DSF) studies. Curves are offset along the vertical axis for clarity. All experiments were performed in triplicate. B. A comparison of the melting temperatures for wild-type A55BB (green), F54E (red), D99A (blue), I48A (orange) and I48E (black) mutants. Upper and lower panels display Tm values for the first and second melting events, respectively. Error bars show the standard errors of the mean from experiments performed in triplicate. C-J. Representative ITC titration curves showing binding of A55BB mutants F54E (C, D), D99A (E, F), I48A (G, H) and I48E (I, J) to Cul3NΔ22 and Cul3N, respectively. Integrated changes in enthalpy (ΔH) are plotted against molar ratio of titrant. The corresponding dissociation constant (KD), number of binding sites (N), enthalpy change (ΔH) and change in Gibbs free energy (ΔG) for each representative experiment are shown. All experiments were performed at least twice independently. Raw data for (C-J) are shown in Fig. S7.

Journal: Journal of Biological Chemistry

Article Title: Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55

doi: 10.1074/jbc.ra118.006561

Figure Lengend Snippet: Figure 6. I48E mutation significantly impairs A55 binding to Cul3. A. Representative thermal melt curves of wild-type A55BB and mutants F54E, D99A, I48A and I48E from differential scanning fluorimetry (DSF) studies. Curves are offset along the vertical axis for clarity. All experiments were performed in triplicate. B. A comparison of the melting temperatures for wild-type A55BB (green), F54E (red), D99A (blue), I48A (orange) and I48E (black) mutants. Upper and lower panels display Tm values for the first and second melting events, respectively. Error bars show the standard errors of the mean from experiments performed in triplicate. C-J. Representative ITC titration curves showing binding of A55BB mutants F54E (C, D), D99A (E, F), I48A (G, H) and I48E (I, J) to Cul3NΔ22 and Cul3N, respectively. Integrated changes in enthalpy (ΔH) are plotted against molar ratio of titrant. The corresponding dissociation constant (KD), number of binding sites (N), enthalpy change (ΔH) and change in Gibbs free energy (ΔG) for each representative experiment are shown. All experiments were performed at least twice independently. Raw data for (C-J) are shown in Fig. S7.

Article Snippet: Human Cul3NΔ22 (Uniprot Q13618, residues 23-388) and Cul3N (Uniprot Q13618, residues 1-388) with the I342R and L346D stabilizing mutations (6) in pNIC-CTHF with C-terminal TEV-cleavable His6-tags were a gift from Nicola BurgessBrown (Addgene plasmids 53672 and 53673).

Techniques: Mutagenesis, Binding Assay, Comparison, Titration