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cul4a (Proteintech)

Name: cul4a
Brand: Proteintech
Rating: 93 (20 reviews)

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Proteintech cul4a

a. Cryo-EM data process of the CSN5i-3-CSN-NEDD8 complex. b. Orientational difference of the CSN5-CSN6 dimer within CSN between the CSN5i-3-CSN-N8∼CRL1 and CSN5i-3-CSN-N8 complex structures. c . A comparison between the binding mode of CSN5i-1a (top panel) and CSN5i-3 (bottom panel) to CSN. Both compounds are shown in spheres. CSN5 is shown in surface representation. NEDD8 bound to CSN5i-3-occupied CSN is shown in gray cartoon. d . The affinity of CSN towards N8∼CRL1 in a saturating amount of CSN5i-1a measured by BLI. e . The affinity of CSN towards N8∼CRL1 in a saturating amount of CSN5i-3 measured by BLI. f . Abundances of <t>CUL4A,</t> DDB1, FBXO22, DCAF4 and FBXL12 in WCE and affinity-purified (AP) CSN2 and CSN6 complexes determined by quantitative MS analyses. g. Assessment of <t>CUL4A,</t> DDB1, and FBXO22 abundances in WCE and affinity-purified CSN2 complexes in the absence and presence of CSN5i-3 using immunoblotting analysis. h . Proteasomal degradation of DDB2 and FBXO22 induced by CSN5i-3.

Cul4a, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Orthosteric Molecular Glue Inhibits COP9 Signalosome with Substrate-Dependent Potency"

Article Title: Orthosteric Molecular Glue Inhibits COP9 Signalosome with Substrate-Dependent Potency

Journal: bioRxiv

doi: 10.1101/2025.11.26.690573

Figure Legend Snippet: a. Cryo-EM data process of the CSN5i-3-CSN-NEDD8 complex. b. Orientational difference of the CSN5-CSN6 dimer within CSN between the CSN5i-3-CSN-N8∼CRL1 and CSN5i-3-CSN-N8 complex structures. c . A comparison between the binding mode of CSN5i-1a (top panel) and CSN5i-3 (bottom panel) to CSN. Both compounds are shown in spheres. CSN5 is shown in surface representation. NEDD8 bound to CSN5i-3-occupied CSN is shown in gray cartoon. d . The affinity of CSN towards N8∼CRL1 in a saturating amount of CSN5i-1a measured by BLI. e . The affinity of CSN towards N8∼CRL1 in a saturating amount of CSN5i-3 measured by BLI. f . Abundances of CUL4A, DDB1, FBXO22, DCAF4 and FBXL12 in WCE and affinity-purified (AP) CSN2 and CSN6 complexes determined by quantitative MS analyses. g. Assessment of CUL4A, DDB1, and FBXO22 abundances in WCE and affinity-purified CSN2 complexes in the absence and presence of CSN5i-3 using immunoblotting analysis. h . Proteasomal degradation of DDB2 and FBXO22 induced by CSN5i-3.

Techniques Used: Cryo-EM Sample Prep, Comparison, Binding Assay, Affinity Purification, Western Blot

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Journal: bioRxiv

Article Title: Orthosteric Molecular Glue Inhibits COP9 Signalosome with Substrate-Dependent Potency

doi: 10.1101/2025.11.26.690573

Figure Lengend Snippet: a. Cryo-EM data process of the CSN5i-3-CSN-NEDD8 complex. b. Orientational difference of the CSN5-CSN6 dimer within CSN between the CSN5i-3-CSN-N8∼CRL1 and CSN5i-3-CSN-N8 complex structures. c . A comparison between the binding mode of CSN5i-1a (top panel) and CSN5i-3 (bottom panel) to CSN. Both compounds are shown in spheres. CSN5 is shown in surface representation. NEDD8 bound to CSN5i-3-occupied CSN is shown in gray cartoon. d . The affinity of CSN towards N8∼CRL1 in a saturating amount of CSN5i-1a measured by BLI. e . The affinity of CSN towards N8∼CRL1 in a saturating amount of CSN5i-3 measured by BLI. f . Abundances of CUL4A, DDB1, FBXO22, DCAF4 and FBXL12 in WCE and affinity-purified (AP) CSN2 and CSN6 complexes determined by quantitative MS analyses. g. Assessment of CUL4A, DDB1, and FBXO22 abundances in WCE and affinity-purified CSN2 complexes in the absence and presence of CSN5i-3 using immunoblotting analysis. h . Proteasomal degradation of DDB2 and FBXO22 induced by CSN5i-3.

Article Snippet: HBTH-CSN2 and HBTH-CSN6 were detected using a streptavidin−HRP conjugate (1:5000), DDB2 was detected by using anti-DDB2 antibody (life technology MA5-34832), Cul4A was detected by using anti-Cul4A (life technology PA5-14542), and FBX022 was detected by using anti-FBX022 (protein tech 13606-1-AP).

Techniques: Cryo-EM Sample Prep, Comparison, Binding Assay, Affinity Purification, Western Blot

Cullin 3, a scaffold protein for Cullin-RING E3 ligases, promotes EV-D68 replication. ( A ) CPEs in EV-D68-infected A549 cells. Cells were treated with MG132 (10 μΜ) or DMSO 36 h after infection with the EV-D68 virus (Fermon, MOI = 0.01). CPEs were observed 48 h post-infection. ( B ) Titers of progeny virions. Supernatants were gathered 48 h post-infection, and viral titers were measured via a standard plaque assay. Data are expressed as the mean ± standard deviation (SD). *** P < 0.001. ( C ) Validation of Cullin family member knockdown efficiency using immunoblotting. ( D ) CPEs after the Cullin family member knockdown in A549 cells. A549 cells pre-transfected with siRNAs were infected with EV-D68 (Fermon, MOI = 0.01). CPEs were observed 48 h post-infection. ( E ) Viral titers of progeny virions in Cullin family member-knockdown A549 cells. Viral titers were determined via a standard plaque assay. Data are expressed as the mean ± SD. ** P < 0.01. n.s ., not significant. ( F through H ) Viral titers in stable Cullin 3-silenced A549 cells. A549 cells were infected with the EV-D68 prototype Fermon (2014) and isolated US/MO/14-18947 (MO) and US/KY/14-18953 (KY) at an MOI of 0.01. Viral titers were determined using a standard plaque assay 48 h post-infection. Data are expressed as the mean ± SD. ** P < 0.01. ( I ) Viral titers in stable Cullin 3-knockdown HEK293T cells. Stable Cullin 3-knockdown or control HEK293T cells were infected with EV-D68 (Fermon, MOI = 0.01). CPEs were observed 48 h post-infection. Data are expressed as the mean ± SD. ** P < 0.01. ( J, K ) Viral titers in Cullin 3-inhibited HEK293T cells. HEK293T cells were transfected with siRNAs or dominant-negative Cullin 3. Wild-type Cullin 3 or an empty vector was co-transfected. Cells of all groups were infected with EV-D68 (Fermon, MOI = 0.01) 24 h post-transfection. Viral titers were determined 48 h post-infection. Data are expressed as the mean ± SD. ** P < 0.01. n.s ., not significant.

Journal: Journal of Virology

Article Title: Cullin 3-mediated ubiquitination restricts enterovirus D68 replication and is counteracted by viral protease 3C

doi: 10.1128/jvi.00354-25

Figure Lengend Snippet: Cullin 3, a scaffold protein for Cullin-RING E3 ligases, promotes EV-D68 replication. ( A ) CPEs in EV-D68-infected A549 cells. Cells were treated with MG132 (10 μΜ) or DMSO 36 h after infection with the EV-D68 virus (Fermon, MOI = 0.01). CPEs were observed 48 h post-infection. ( B ) Titers of progeny virions. Supernatants were gathered 48 h post-infection, and viral titers were measured via a standard plaque assay. Data are expressed as the mean ± standard deviation (SD). *** P < 0.001. ( C ) Validation of Cullin family member knockdown efficiency using immunoblotting. ( D ) CPEs after the Cullin family member knockdown in A549 cells. A549 cells pre-transfected with siRNAs were infected with EV-D68 (Fermon, MOI = 0.01). CPEs were observed 48 h post-infection. ( E ) Viral titers of progeny virions in Cullin family member-knockdown A549 cells. Viral titers were determined via a standard plaque assay. Data are expressed as the mean ± SD. ** P < 0.01. n.s ., not significant. ( F through H ) Viral titers in stable Cullin 3-silenced A549 cells. A549 cells were infected with the EV-D68 prototype Fermon (2014) and isolated US/MO/14-18947 (MO) and US/KY/14-18953 (KY) at an MOI of 0.01. Viral titers were determined using a standard plaque assay 48 h post-infection. Data are expressed as the mean ± SD. ** P < 0.01. ( I ) Viral titers in stable Cullin 3-knockdown HEK293T cells. Stable Cullin 3-knockdown or control HEK293T cells were infected with EV-D68 (Fermon, MOI = 0.01). CPEs were observed 48 h post-infection. Data are expressed as the mean ± SD. ** P < 0.01. ( J, K ) Viral titers in Cullin 3-inhibited HEK293T cells. HEK293T cells were transfected with siRNAs or dominant-negative Cullin 3. Wild-type Cullin 3 or an empty vector was co-transfected. Cells of all groups were infected with EV-D68 (Fermon, MOI = 0.01) 24 h post-transfection. Viral titers were determined 48 h post-infection. Data are expressed as the mean ± SD. ** P < 0.01. n.s ., not significant.

Article Snippet: The following antibodies were used: anti-Cullin 1 antibody (Abcam, ab75817), anti-Cullin 2 antibody (Abways, CY1025), anti-Cullin 3 antibody (Proteintech, 11107-1-AP), anti-Cullin 4A antibody (Proteintech, 10693-1-AP), anti-Cullin 4B antibody (Proteintech, 12916-1-AP), anti-Cullin 5 antibody (arigo, ARG59155 ), anti-EV-D68-VP1 antibody (GeneTex, GTX132313), anti-HA-tag antibody (Thermo Fisher, 71-5500), anti-Ubiquitin antibody (Proteintech, 10201-2-AP), anti-myc-tag antibody (Sigma, M5546), anti-α-Tubulin antibody (GenScript, A01410), and anti-histone H3 antibody (Abcam, ab176842).

Techniques: Infection, Virus, Plaque Assay, Standard Deviation, Biomarker Discovery, Knockdown, Western Blot, Transfection, Isolation, Control, Dominant Negative Mutation, Plasmid Preparation

Cullin 3 negatively regulates EV-D68 VP1 protein level. ( A and B ) Virus attachment and entry assays. Stable Cullin 3-knockdown A549 cells were incubated with EV-D68 (Fermon, MOI = 1) at 4 ℃ or 37 ℃ for 2 h, and cells were washed with DMEM to remove unbound viruses. Viral RNA was quantified using qRT-PCR. Data are expressed as the mean ± SD. n.s ., not significant. ( C and D ) EV-D68 5′ UTR activity. Plasmids with the EV-D68 5′ UTR were transfected into HEK293T cells, along with dominant-negative Cullin 3 ( C ) or wild-type Cullin 3 ( D ) plasmids. Control groups were transfected with a control vector. Renilla luciferase activity served as an internal control. The activities of Firefly and Renilla luciferases were measured 48 h post-transfection. The bar graph represents the ratio of Firefly and Renilla luciferase. Data are expressed as the mean ± SD. n.s ., not significant. ( E ) EV-D68 VP1 levels in Cullin 3-knockdown A549 cells. A549 cells pre-transfected with the indicated siRNAs were infected with EV-D68 (Fermon, MOI = 0.05). Cells were harvested 2, 4, 6, 8, and 10 h after infection. VP1 expression levels were detected using an immunoblotting assay. ( F ) VP1–VP3 protein levels in Cullin 3-inhibited HEK293T cells. HEK293T cells were transfected with dominant-negative Cullin 3 or control vectors and VP1–VP3 plasmids. VP1–VP3 levels were measured 48 h after transfection. ( G ) The abundance of VP1–VP3 was quantified using ImageJ software. Data are expressed as the mean ± SD. **** P < 0.0001. *** P < 0.001. ** P < 0.01. ( H ) Immunoblot analysis of VP1 level. HEK293T cells pre-transfected with the VP1 plasmids were treated with MG132 (10 μΜ), MLN4924 (2 μΜ), CQ (50 μΜ), or 3-MA (5 mΜ). The VP1 levels were measured 12 h after treatment.

Journal: Journal of Virology

Article Title: Cullin 3-mediated ubiquitination restricts enterovirus D68 replication and is counteracted by viral protease 3C

doi: 10.1128/jvi.00354-25

Figure Lengend Snippet: Cullin 3 negatively regulates EV-D68 VP1 protein level. ( A and B ) Virus attachment and entry assays. Stable Cullin 3-knockdown A549 cells were incubated with EV-D68 (Fermon, MOI = 1) at 4 ℃ or 37 ℃ for 2 h, and cells were washed with DMEM to remove unbound viruses. Viral RNA was quantified using qRT-PCR. Data are expressed as the mean ± SD. n.s ., not significant. ( C and D ) EV-D68 5′ UTR activity. Plasmids with the EV-D68 5′ UTR were transfected into HEK293T cells, along with dominant-negative Cullin 3 ( C ) or wild-type Cullin 3 ( D ) plasmids. Control groups were transfected with a control vector. Renilla luciferase activity served as an internal control. The activities of Firefly and Renilla luciferases were measured 48 h post-transfection. The bar graph represents the ratio of Firefly and Renilla luciferase. Data are expressed as the mean ± SD. n.s ., not significant. ( E ) EV-D68 VP1 levels in Cullin 3-knockdown A549 cells. A549 cells pre-transfected with the indicated siRNAs were infected with EV-D68 (Fermon, MOI = 0.05). Cells were harvested 2, 4, 6, 8, and 10 h after infection. VP1 expression levels were detected using an immunoblotting assay. ( F ) VP1–VP3 protein levels in Cullin 3-inhibited HEK293T cells. HEK293T cells were transfected with dominant-negative Cullin 3 or control vectors and VP1–VP3 plasmids. VP1–VP3 levels were measured 48 h after transfection. ( G ) The abundance of VP1–VP3 was quantified using ImageJ software. Data are expressed as the mean ± SD. **** P < 0.0001. *** P < 0.001. ** P < 0.01. ( H ) Immunoblot analysis of VP1 level. HEK293T cells pre-transfected with the VP1 plasmids were treated with MG132 (10 μΜ), MLN4924 (2 μΜ), CQ (50 μΜ), or 3-MA (5 mΜ). The VP1 levels were measured 12 h after treatment.

Techniques: Virus, Knockdown, Incubation, Quantitative RT-PCR, Activity Assay, Transfection, Dominant Negative Mutation, Control, Plasmid Preparation, Luciferase, Infection, Expressing, Western Blot, Software

Cullin 3 promotes EV-D68 VP1 ubiquitination. ( A ) Analysis of VP1 in Cullin 3-pull-down precipitates. HEK293T cells pre-transfected with Cullin 3 or empty control constructs were infected with EV-D68 (Fermon, MOI = 0.01). Cells were harvested 48 h post-infection and analyzed via co-IP and immunoblotting. ( B ) Analysis of VP1 in Cullin 3-pull-down precipitates. HEK293T cells pre-transfected with Cullin 3 or empty control constructs, along with VP1-expressing vectors, were harvested 48 h post-transfection and analyzed using co-IP and immunoblotting. ( C ) Subcellular localization of Cullin 3 and VP1. HEK293T cells were transfected with Cullin 3 and VP1 plasmids for 48 h. The cells were then analyzed by performing immunofluorescence and confocal microscopy. Scale bar, 10 µm. ( D ) VP1 ubiquitination analysis. HEK293T cells pre-transfected with siRNAs were transfected with EV-D68 VP1 or empty control vectors. Cells were treated with MG132 (10 μΜ) for 12 h before harvesting and lysis.

Journal: Journal of Virology

Article Title: Cullin 3-mediated ubiquitination restricts enterovirus D68 replication and is counteracted by viral protease 3C

doi: 10.1128/jvi.00354-25

Figure Lengend Snippet: Cullin 3 promotes EV-D68 VP1 ubiquitination. ( A ) Analysis of VP1 in Cullin 3-pull-down precipitates. HEK293T cells pre-transfected with Cullin 3 or empty control constructs were infected with EV-D68 (Fermon, MOI = 0.01). Cells were harvested 48 h post-infection and analyzed via co-IP and immunoblotting. ( B ) Analysis of VP1 in Cullin 3-pull-down precipitates. HEK293T cells pre-transfected with Cullin 3 or empty control constructs, along with VP1-expressing vectors, were harvested 48 h post-transfection and analyzed using co-IP and immunoblotting. ( C ) Subcellular localization of Cullin 3 and VP1. HEK293T cells were transfected with Cullin 3 and VP1 plasmids for 48 h. The cells were then analyzed by performing immunofluorescence and confocal microscopy. Scale bar, 10 µm. ( D ) VP1 ubiquitination analysis. HEK293T cells pre-transfected with siRNAs were transfected with EV-D68 VP1 or empty control vectors. Cells were treated with MG132 (10 μΜ) for 12 h before harvesting and lysis.

Techniques: Ubiquitin Proteomics, Transfection, Control, Construct, Infection, Co-Immunoprecipitation Assay, Western Blot, Expressing, Immunofluorescence, Confocal Microscopy, Lysis

EV-D68 infection triggers the cleavage of Cullin 3 via the enzymatic activity of 3C. ( A, B ) Analysis of the host Cullin 3 level. A549 cells were infected with the EV-D68 prototype Fermon (2014) ( A ) or the isolated US/MO/14-18947 (MO) and US/KY/14-18953 (KY) ( B ) at an MOI of 0.01. Cullin 3 was detected via immunoblotting 48 h post-infection. ( C and D ) Immunoblotting analysis of Cullin 3. HEK293T and RD cells were infected with EV-D68 (Fermon, MOI = 0.01). Cullin 3 was detected 48 h post-infection. ( E ) Screening of Cullin 3 cleavage by EV-D68-encoded proteins. HEK293T cells were transfected with myc-tagged Cullin 3-expressing vectors and indicated EV-D68-encoded proteins. Cell lysates were analyzed 48 h post-transfection.

Journal: Journal of Virology

Article Title: Cullin 3-mediated ubiquitination restricts enterovirus D68 replication and is counteracted by viral protease 3C

doi: 10.1128/jvi.00354-25

Figure Lengend Snippet: EV-D68 infection triggers the cleavage of Cullin 3 via the enzymatic activity of 3C. ( A, B ) Analysis of the host Cullin 3 level. A549 cells were infected with the EV-D68 prototype Fermon (2014) ( A ) or the isolated US/MO/14-18947 (MO) and US/KY/14-18953 (KY) ( B ) at an MOI of 0.01. Cullin 3 was detected via immunoblotting 48 h post-infection. ( C and D ) Immunoblotting analysis of Cullin 3. HEK293T and RD cells were infected with EV-D68 (Fermon, MOI = 0.01). Cullin 3 was detected 48 h post-infection. ( E ) Screening of Cullin 3 cleavage by EV-D68-encoded proteins. HEK293T cells were transfected with myc-tagged Cullin 3-expressing vectors and indicated EV-D68-encoded proteins. Cell lysates were analyzed 48 h post-transfection.

Techniques: Infection, Activity Assay, Isolation, Western Blot, Transfection, Expressing

Enterovirus 3C with conserved enzyme activity sites induces Cullin 3 cleavage. ( A ) Cullin 3 cleavage treatment with zVAD. HEK293T cells transfected with Cullin 3 and EV-D68 3C plasmids were cultured in a medium containing Z-VAD-FMK (20 µmol/L) for 48 h. ( B ) Structure of EV-D68 3C protease. ( C ) Sequence alignment of the enteroviral 3C proteases. H40, E71, and C147 indicate the conserved enzyme activity sites. ( D ) Cullin 3 cleavage by 3C protease-defective mutants. ( E ) Relative intensities of cleaved Cullin 3 were quantified using ImageJ software. Data are expressed as the mean ± SD. ** P < 0.01. ( F ) Cullin 3 cleavage treatment with GC376. HEK293T cells pre-transfected with Cullin 3 and EV-D68 3C vectors were cultured in a medium containing GC376 (1 µmol/L) for 24 h. ( G ) Cullin 3 cleavage by the indicated enterovirus 3C proteases. ( H ) Relative abundances of cleaved Cullin 3, quantified using ImageJ software. Data are expressed as the mean ± SD. ** P < 0.01.

Journal: Journal of Virology

Article Title: Cullin 3-mediated ubiquitination restricts enterovirus D68 replication and is counteracted by viral protease 3C

doi: 10.1128/jvi.00354-25

Figure Lengend Snippet: Enterovirus 3C with conserved enzyme activity sites induces Cullin 3 cleavage. ( A ) Cullin 3 cleavage treatment with zVAD. HEK293T cells transfected with Cullin 3 and EV-D68 3C plasmids were cultured in a medium containing Z-VAD-FMK (20 µmol/L) for 48 h. ( B ) Structure of EV-D68 3C protease. ( C ) Sequence alignment of the enteroviral 3C proteases. H40, E71, and C147 indicate the conserved enzyme activity sites. ( D ) Cullin 3 cleavage by 3C protease-defective mutants. ( E ) Relative intensities of cleaved Cullin 3 were quantified using ImageJ software. Data are expressed as the mean ± SD. ** P < 0.01. ( F ) Cullin 3 cleavage treatment with GC376. HEK293T cells pre-transfected with Cullin 3 and EV-D68 3C vectors were cultured in a medium containing GC376 (1 µmol/L) for 24 h. ( G ) Cullin 3 cleavage by the indicated enterovirus 3C proteases. ( H ) Relative abundances of cleaved Cullin 3, quantified using ImageJ software. Data are expressed as the mean ± SD. ** P < 0.01.

Techniques: Activity Assay, Transfection, Cell Culture, Sequencing, Software

Journal: Journal of Virology

Article Title: Cullin 3-mediated ubiquitination restricts enterovirus D68 replication and is counteracted by viral protease 3C

doi: 10.1128/jvi.00354-25

Figure Lengend Snippet: EV-D68 cleaves Cullin 3 at the Q681 residue. ( A ) Sequence logo analysis of the predicted EV-D68 3C protease cleavage site. ( B ) Cullin 3 mutants cleaved by EV-D68 3C. HEK293T cells were transfected with EV-D68 3C and the indicated myc-tagged Cullin 3 plasmids. Cells were harvested 48 h post-transfection. Cullin 3 was detected via immunoblotting. ( C ) Relative densities of cleaved Cullin 3 quantified using ImageJ software. Data are expressed as the mean ± SD. *** P < 0.001. ( D ) Subcellular localization of Cullin 3 and EV-D68 3C. HEK293T cells pre-transfected with Cullin 3 and 3C plasmids were cultured for 48 h. The subcellular localization of these proteins was examined by performing immunofluorescence and confocal microscopy. Scale bar, 10 µm.

Techniques: Residue, Sequencing, Transfection, Western Blot, Software, Cell Culture, Immunofluorescence, Confocal Microscopy

Cullin 3 cleavage disrupts VP1 ubiquitination. ( A ) Immunoblotting analysis of Cullin 3-mediated ubiquitination. HEK293T cells were transfected with the indicated vectors. Whole-cell lysates and myc pull-down products were obtained from HEK293T cell lysates. ( B ) Analysis of Cullin 3 and its cleaved products in VP1-pull-down precipitates. HEK293T cells were transfected with the indicated vectors. Whole-cell lysates and HA pull-down products were analyzed. ( C ) Working model illustrating how VP1 interacts with Cullin 3.

Journal: Journal of Virology

Article Title: Cullin 3-mediated ubiquitination restricts enterovirus D68 replication and is counteracted by viral protease 3C

doi: 10.1128/jvi.00354-25

Figure Lengend Snippet: Cullin 3 cleavage disrupts VP1 ubiquitination. ( A ) Immunoblotting analysis of Cullin 3-mediated ubiquitination. HEK293T cells were transfected with the indicated vectors. Whole-cell lysates and myc pull-down products were obtained from HEK293T cell lysates. ( B ) Analysis of Cullin 3 and its cleaved products in VP1-pull-down precipitates. HEK293T cells were transfected with the indicated vectors. Whole-cell lysates and HA pull-down products were analyzed. ( C ) Working model illustrating how VP1 interacts with Cullin 3.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection

EV-D68 3C suppresses L1 mobilization via Cullin 3 cleavage. ( A ) Schematic representation of EGFP reporter-based retrotransposition assay. A positive signal can only be detected after L1 has been transcribed, spliced, translated, reverse-transcribed, and integrated into the host genome. ( B ) Schematic of LINE-1 plasmids. ( C, D ) L1 retrotransposition mediated by Cullin 3 overexpression, as determined via flow cytometry. HEK293T cells were transfected with L1RP EGFP plasmids, along with Cullin 3 or an empty vector. Transfected cells were selected with puromycin (3 µg/mL for 2 days) 48 h post-transfection. The percentage of GFP (+) cells was measured using flow cytometry. The data are expressed as the mean ± SD. ** P < 0.01. ( E, F ) L1 mobility following Cullin 3 knockdown based on flow cytometry. Data are expressed as the mean ± SD. *** P < 0.001. ( G, H ) L1 mobility was determined using a dual luciferase assay. HEK293T cells were transfected with pYX014 or pYX017 plasmids, and L1 mobility was measured using a dual-luciferase assay after 4 days. Data are expressed as the mean ± SD. ** P < 0.01. ( I ) L1 mobilization mediated by the EV-D68 3C protease. ( J ) L1 transposition in EV-D68 3C-overexpressing HEK293T cells. HEK293T cells were transfected with L1RP EGFP plasmids, along with EV-D68 3C or an empty vector. Cullin 3 or an empty vector was co-transfected. L1 mobility was measured using flow cytometry after 4 days. The data are expressed as the mean ± SD. * P < 0.1. **** P < 0.0001. ( K ) L1 mobility in Cullin 3-knockdown HEK293T cells. HEK293T cells were transfected with siCullin 3 and pYX014. Cullin 3, Cullin 3 (1–681 aa), Cullin 3 (682–768 aa), or an empty vector were co-transfected. L1 mobility was measured using a dual-luciferase assay after 4 days. The data are expressed as the mean ± SD. ** P < 0.01. n.s ., not significant.

Journal: Journal of Virology

Article Title: Cullin 3-mediated ubiquitination restricts enterovirus D68 replication and is counteracted by viral protease 3C

doi: 10.1128/jvi.00354-25

Figure Lengend Snippet: EV-D68 3C suppresses L1 mobilization via Cullin 3 cleavage. ( A ) Schematic representation of EGFP reporter-based retrotransposition assay. A positive signal can only be detected after L1 has been transcribed, spliced, translated, reverse-transcribed, and integrated into the host genome. ( B ) Schematic of LINE-1 plasmids. ( C, D ) L1 retrotransposition mediated by Cullin 3 overexpression, as determined via flow cytometry. HEK293T cells were transfected with L1RP EGFP plasmids, along with Cullin 3 or an empty vector. Transfected cells were selected with puromycin (3 µg/mL for 2 days) 48 h post-transfection. The percentage of GFP (+) cells was measured using flow cytometry. The data are expressed as the mean ± SD. ** P < 0.01. ( E, F ) L1 mobility following Cullin 3 knockdown based on flow cytometry. Data are expressed as the mean ± SD. *** P < 0.001. ( G, H ) L1 mobility was determined using a dual luciferase assay. HEK293T cells were transfected with pYX014 or pYX017 plasmids, and L1 mobility was measured using a dual-luciferase assay after 4 days. Data are expressed as the mean ± SD. ** P < 0.01. ( I ) L1 mobilization mediated by the EV-D68 3C protease. ( J ) L1 transposition in EV-D68 3C-overexpressing HEK293T cells. HEK293T cells were transfected with L1RP EGFP plasmids, along with EV-D68 3C or an empty vector. Cullin 3 or an empty vector was co-transfected. L1 mobility was measured using flow cytometry after 4 days. The data are expressed as the mean ± SD. * P < 0.1. **** P < 0.0001. ( K ) L1 mobility in Cullin 3-knockdown HEK293T cells. HEK293T cells were transfected with siCullin 3 and pYX014. Cullin 3, Cullin 3 (1–681 aa), Cullin 3 (682–768 aa), or an empty vector were co-transfected. L1 mobility was measured using a dual-luciferase assay after 4 days. The data are expressed as the mean ± SD. ** P < 0.01. n.s ., not significant.

Techniques: Reverse Transcription, Over Expression, Flow Cytometry, Transfection, Plasmid Preparation, Knockdown, Luciferase

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