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ubch5a e2  (R&D Systems)


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    R&D Systems ubch5a e2
    Ubch5a E2, supplied by R&D Systems, used in various techniques. Bioz Stars score: 95/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ubch5a/Recombinant+Human+UbcH5a%2FUBE2D1+Protein%2C+CF/us12590953-830-6-15
    Average 95 stars, based on 77 article reviews
    ubch5a e2 - by Bioz Stars, 2026-09
    95/100 stars

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    Related Articles

    Recombinant:

    Article Title: Ubiquitylation of nucleic acids by DELTEX ubiquitin E3 ligase DTX3L.
    Article Snippet: .. UBE1 (E-304-050), UBCH5A (E2-616-100), and recombinant Ub (U-100H-10M) were purchased from R&D Systems. .. Exo T nuclease (M0265S) was purchased from NEB.

    Article Title: The CRL3 KCTD10 ubiquitin ligase–USP18 axis coordinately regulates cystine uptake and ferroptosis by modulating SLC7A11
    Article Snippet: Antibodies and reagents The following antibodies were used: β-actin (Sigma-Aldrich, A5441, dilution: 1:10000); CUL-1 (Santa Cruz, sc-11384, dilution: 1:1000); CUL-2 (Abcam, ab166917, dilution: 1:1000); CUL-3 (Cell Signaling Technology, 2759S, dilution: 1:1000); CUL-4A (Cell Signaling Technology, 2699S, dilution: 1:1000); CUL-4B (Proteintech, 12916-1-AP, dilution: 1:1000); CUL-5 (Santa Cruz, sc-13014, dilution: 1:1000); FLAG, clone M2 (Sigma-Aldrich, F1804-500UG, dilution: 1:2000); DUBA (Proteintech, 21002-1-AP, dilution: 1:1000); FLAG M2 affinity gel (Sigma-Aldrich, A2220-5ML); 3ÎFLAG peptide (MCE, HY-P0319A); HA (Sigma, H6908, dilution: 1:2000); Anti-HA High Affinity (3F10) (Roche, 11867423001); KCTD10 (Proteintech, 27279-1-AP, dilution: 1:1000); KCTD10 (Sigma-Aldrich, HPA014273, dilution: 1:1000); KLHL13 (Proteintech, 15612-1-AP, dilution: 1:1000); KLHL25 (Proteintech, 25066-1-AP, dilution: 1:1000); NAEβ (Abcam, ab124728, dilution: 1:1000); USP18 (Cell Signaling Technology, D4E7, dilution: 1:1000); USP18 (ABclonal, A16739, dilution: 1:1000); Recombinant Anti-Sodium Potassium ATPase antibody (Abcam, ab76020); SLC3A2 (Proteintech, 15193-1-AP, dilution: 1:10000); SLC7A11 (Cell Signaling Technology, D2M7A, dilution: 1:1000); SLC7A11 (Novus, NB300-318, dilution: 1:1000); SOCS2 (Signalway Antiboday, 49110, dilution: 1:1000); TRIM26 (Proteintech, 27013-1-AP, dilution: 1:1000); α-tubulin (Sigma, Clone AA13, T8203, dilution: 1:10000); OTUB1 (ABclonal, A11656, dilution: 1:1000); Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson, 11-035-144, dilution: 1:4000); Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson, 115-035-146, dilution: 1:4000); Peroxidase AffiniPure Goat Anti-Rat IgG (H+L) (Jackson, 112-035-143, dilution: 1:4000);Ubiquitin (Santa Cruz, P4D1, sc-8017, dilution: 1:1000); ZRANB1 (Abcam, ab103417, dilution: 1:1000) was gift from Dr. Peijing Zhang (Huazhong University of Science and Technology, China). .. Reagents were obtained from the following suppliers: Chlorhexidine (CHX) (Sigma, C7698); Ferrostatin-1 (APExBIO, A4371); MG132 (MedChem Express, HY-13259); Erastin (Selleck, S7242); HPBCD (Topscience, T27563); IKE (Selleck, S8877); Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, 13778-150); Lipofectamine 3000 Transfection Reagent (Invitrogen, L3000-015); Ni-NTA Agarose (Qiagen, 30210); MLN4924 (ApexBio, B1036); Puromycin (VWR Life Science, 0336C120); Recombinant Human Ubiquitin Activating Enzyme (UBE1) (R&D Systems, E-305-025); Recombinant Human Ubiquitin Conjugating Enzyme His6-Cdc34/UbcH3 (R&D Systems, E2-610-100) and UbcH5a (R&D Systems, E2-616-100); Recombinant Human Ubiquitin Protein (U-100H-10M); RSL3 (Topscience, T3646). .. A variety of USP plasmids were gifts from Dr. Lingqiang Zhang (Beijing Institute of Lifeomics, China) and USP18 truncated plasmids were gifts from Dr. Bo Zhong (Wuhan University, China).

    Article Title: PHD3-VHL axis controls HIV-2 infection through oxygen-dependent hydroxylation and degradation of Vpx.
    Article Snippet: HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit, Cell Signaling) were used at 1:5000 dilution. .. In vitro ubiquitination reactions (10 μL) contained 1 μg recombinant HIV-2 GH-1 Vpx or P41A mutant (synthesized using wheat germ cell-free protein synthesis system), 100 nM ubiquitin-activating enzyme E1 (Merck, #SRP6147), 1 μM UbcH5a (R&D Systems, #E2-616), 100 nM EloB/EloC/VHL/CUL2/RBX1 (R&D Systems, #E3-655–025), 5 μM ubiquitin (Merck, #U5507), 2.5 mM MgATP solution (R&D Systems, #B-20), and 1x E3 Ligase Reaction buffer (R&D Systems, #B-71). .. For PHD3-dependent ubiquitination assays, Vpx was pre-incubated with 300 nM recombinant PHD3/EGLN3 protein (Active Motif, #81033) in PHD3 reaction buffer (20 mM Tris-HCl pH 7.5, 5 mM KCl, 1.5 mM MgCl 2 , 1 mM DTT, 100 μM 2-oxoglutarate, 100 μM ascorbate, and 50 μM (NH 4 ) 2 Fe(SO 4 ) 2 ·6H 2 O) at 30°C for 60 min.

    Article Title: DELTEX E3 ligases ubiquitylate ADP-ribosyl modification on nucleic acids.
    Article Snippet: .. NUDT16 ( ab103059 ) was purchased from Abcam; UBE1 ( E-304-050 ) , UBCH5A ( E2-616-100 ) , and recombinant Ub ( WT and G76A; U-100H-10M and UM-G76A-100, respectively ) were purchased from R&D Systems. ..

    Article Title: PHD3-VHL axis controls HIV-2 infection through oxygen-dependent hydroxylation and degradation of Vpx
    Article Snippet: HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit, Cell Signaling) were used at 1:5000 dilution. .. In vitro ubiquitination reactions (10 μL) contained 1 μg recombinant HIV-2 GH-1 Vpx or P41A mutant (synthesized using wheat germ cell-free protein synthesis system), 100 nM ubiquitin-activating enzyme E1 (Merck, #SRP6147), 1 μM UbcH5a (R&D Systems, #E2-616), 100 nM EloB/EloC/VHL/CUL2/RBX1 (R&D Systems, #E3-655–025), 5 μM ubiquitin (Merck, #U5507), 2.5 mM MgATP solution (R&D Systems, #B-20), and 1x E3 Ligase Reaction buffer (R&D Systems, #B-71). .. For PHD3-dependent ubiquitination assays, Vpx was pre-incubated with 300 nM recombinant PHD3/EGLN3 protein (Active Motif, #81033) in PHD3 reaction buffer (20 mM Tris-HCl pH 7.5, 5 mM KCl, 1.5 mM MgCl 2 , 1 mM DTT, 100 μM 2-oxoglutarate, 100 μM ascorbate, and 50 μM (NH 4 ) 2 Fe(SO 4 ) 2 ·6H 2 O) at 30°C for 60 min.

    Transfection:

    Article Title: The CRL3 KCTD10 ubiquitin ligase–USP18 axis coordinately regulates cystine uptake and ferroptosis by modulating SLC7A11
    Article Snippet: Antibodies and reagents The following antibodies were used: β-actin (Sigma-Aldrich, A5441, dilution: 1:10000); CUL-1 (Santa Cruz, sc-11384, dilution: 1:1000); CUL-2 (Abcam, ab166917, dilution: 1:1000); CUL-3 (Cell Signaling Technology, 2759S, dilution: 1:1000); CUL-4A (Cell Signaling Technology, 2699S, dilution: 1:1000); CUL-4B (Proteintech, 12916-1-AP, dilution: 1:1000); CUL-5 (Santa Cruz, sc-13014, dilution: 1:1000); FLAG, clone M2 (Sigma-Aldrich, F1804-500UG, dilution: 1:2000); DUBA (Proteintech, 21002-1-AP, dilution: 1:1000); FLAG M2 affinity gel (Sigma-Aldrich, A2220-5ML); 3ÎFLAG peptide (MCE, HY-P0319A); HA (Sigma, H6908, dilution: 1:2000); Anti-HA High Affinity (3F10) (Roche, 11867423001); KCTD10 (Proteintech, 27279-1-AP, dilution: 1:1000); KCTD10 (Sigma-Aldrich, HPA014273, dilution: 1:1000); KLHL13 (Proteintech, 15612-1-AP, dilution: 1:1000); KLHL25 (Proteintech, 25066-1-AP, dilution: 1:1000); NAEβ (Abcam, ab124728, dilution: 1:1000); USP18 (Cell Signaling Technology, D4E7, dilution: 1:1000); USP18 (ABclonal, A16739, dilution: 1:1000); Recombinant Anti-Sodium Potassium ATPase antibody (Abcam, ab76020); SLC3A2 (Proteintech, 15193-1-AP, dilution: 1:10000); SLC7A11 (Cell Signaling Technology, D2M7A, dilution: 1:1000); SLC7A11 (Novus, NB300-318, dilution: 1:1000); SOCS2 (Signalway Antiboday, 49110, dilution: 1:1000); TRIM26 (Proteintech, 27013-1-AP, dilution: 1:1000); α-tubulin (Sigma, Clone AA13, T8203, dilution: 1:10000); OTUB1 (ABclonal, A11656, dilution: 1:1000); Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson, 11-035-144, dilution: 1:4000); Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson, 115-035-146, dilution: 1:4000); Peroxidase AffiniPure Goat Anti-Rat IgG (H+L) (Jackson, 112-035-143, dilution: 1:4000);Ubiquitin (Santa Cruz, P4D1, sc-8017, dilution: 1:1000); ZRANB1 (Abcam, ab103417, dilution: 1:1000) was gift from Dr. Peijing Zhang (Huazhong University of Science and Technology, China). .. Reagents were obtained from the following suppliers: Chlorhexidine (CHX) (Sigma, C7698); Ferrostatin-1 (APExBIO, A4371); MG132 (MedChem Express, HY-13259); Erastin (Selleck, S7242); HPBCD (Topscience, T27563); IKE (Selleck, S8877); Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, 13778-150); Lipofectamine 3000 Transfection Reagent (Invitrogen, L3000-015); Ni-NTA Agarose (Qiagen, 30210); MLN4924 (ApexBio, B1036); Puromycin (VWR Life Science, 0336C120); Recombinant Human Ubiquitin Activating Enzyme (UBE1) (R&D Systems, E-305-025); Recombinant Human Ubiquitin Conjugating Enzyme His6-Cdc34/UbcH3 (R&D Systems, E2-610-100) and UbcH5a (R&D Systems, E2-616-100); Recombinant Human Ubiquitin Protein (U-100H-10M); RSL3 (Topscience, T3646). .. A variety of USP plasmids were gifts from Dr. Lingqiang Zhang (Beijing Institute of Lifeomics, China) and USP18 truncated plasmids were gifts from Dr. Bo Zhong (Wuhan University, China).

    Ubiquitin Proteomics:

    Article Title: The CRL3 KCTD10 ubiquitin ligase–USP18 axis coordinately regulates cystine uptake and ferroptosis by modulating SLC7A11
    Article Snippet: Antibodies and reagents The following antibodies were used: β-actin (Sigma-Aldrich, A5441, dilution: 1:10000); CUL-1 (Santa Cruz, sc-11384, dilution: 1:1000); CUL-2 (Abcam, ab166917, dilution: 1:1000); CUL-3 (Cell Signaling Technology, 2759S, dilution: 1:1000); CUL-4A (Cell Signaling Technology, 2699S, dilution: 1:1000); CUL-4B (Proteintech, 12916-1-AP, dilution: 1:1000); CUL-5 (Santa Cruz, sc-13014, dilution: 1:1000); FLAG, clone M2 (Sigma-Aldrich, F1804-500UG, dilution: 1:2000); DUBA (Proteintech, 21002-1-AP, dilution: 1:1000); FLAG M2 affinity gel (Sigma-Aldrich, A2220-5ML); 3ÎFLAG peptide (MCE, HY-P0319A); HA (Sigma, H6908, dilution: 1:2000); Anti-HA High Affinity (3F10) (Roche, 11867423001); KCTD10 (Proteintech, 27279-1-AP, dilution: 1:1000); KCTD10 (Sigma-Aldrich, HPA014273, dilution: 1:1000); KLHL13 (Proteintech, 15612-1-AP, dilution: 1:1000); KLHL25 (Proteintech, 25066-1-AP, dilution: 1:1000); NAEβ (Abcam, ab124728, dilution: 1:1000); USP18 (Cell Signaling Technology, D4E7, dilution: 1:1000); USP18 (ABclonal, A16739, dilution: 1:1000); Recombinant Anti-Sodium Potassium ATPase antibody (Abcam, ab76020); SLC3A2 (Proteintech, 15193-1-AP, dilution: 1:10000); SLC7A11 (Cell Signaling Technology, D2M7A, dilution: 1:1000); SLC7A11 (Novus, NB300-318, dilution: 1:1000); SOCS2 (Signalway Antiboday, 49110, dilution: 1:1000); TRIM26 (Proteintech, 27013-1-AP, dilution: 1:1000); α-tubulin (Sigma, Clone AA13, T8203, dilution: 1:10000); OTUB1 (ABclonal, A11656, dilution: 1:1000); Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson, 11-035-144, dilution: 1:4000); Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson, 115-035-146, dilution: 1:4000); Peroxidase AffiniPure Goat Anti-Rat IgG (H+L) (Jackson, 112-035-143, dilution: 1:4000);Ubiquitin (Santa Cruz, P4D1, sc-8017, dilution: 1:1000); ZRANB1 (Abcam, ab103417, dilution: 1:1000) was gift from Dr. Peijing Zhang (Huazhong University of Science and Technology, China). .. Reagents were obtained from the following suppliers: Chlorhexidine (CHX) (Sigma, C7698); Ferrostatin-1 (APExBIO, A4371); MG132 (MedChem Express, HY-13259); Erastin (Selleck, S7242); HPBCD (Topscience, T27563); IKE (Selleck, S8877); Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, 13778-150); Lipofectamine 3000 Transfection Reagent (Invitrogen, L3000-015); Ni-NTA Agarose (Qiagen, 30210); MLN4924 (ApexBio, B1036); Puromycin (VWR Life Science, 0336C120); Recombinant Human Ubiquitin Activating Enzyme (UBE1) (R&D Systems, E-305-025); Recombinant Human Ubiquitin Conjugating Enzyme His6-Cdc34/UbcH3 (R&D Systems, E2-610-100) and UbcH5a (R&D Systems, E2-616-100); Recombinant Human Ubiquitin Protein (U-100H-10M); RSL3 (Topscience, T3646). .. A variety of USP plasmids were gifts from Dr. Lingqiang Zhang (Beijing Institute of Lifeomics, China) and USP18 truncated plasmids were gifts from Dr. Bo Zhong (Wuhan University, China).

    Article Title: PHD3-VHL axis controls HIV-2 infection through oxygen-dependent hydroxylation and degradation of Vpx.
    Article Snippet: HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit, Cell Signaling) were used at 1:5000 dilution. .. In vitro ubiquitination reactions (10 μL) contained 1 μg recombinant HIV-2 GH-1 Vpx or P41A mutant (synthesized using wheat germ cell-free protein synthesis system), 100 nM ubiquitin-activating enzyme E1 (Merck, #SRP6147), 1 μM UbcH5a (R&D Systems, #E2-616), 100 nM EloB/EloC/VHL/CUL2/RBX1 (R&D Systems, #E3-655–025), 5 μM ubiquitin (Merck, #U5507), 2.5 mM MgATP solution (R&D Systems, #B-20), and 1x E3 Ligase Reaction buffer (R&D Systems, #B-71). .. For PHD3-dependent ubiquitination assays, Vpx was pre-incubated with 300 nM recombinant PHD3/EGLN3 protein (Active Motif, #81033) in PHD3 reaction buffer (20 mM Tris-HCl pH 7.5, 5 mM KCl, 1.5 mM MgCl 2 , 1 mM DTT, 100 μM 2-oxoglutarate, 100 μM ascorbate, and 50 μM (NH 4 ) 2 Fe(SO 4 ) 2 ·6H 2 O) at 30°C for 60 min.

    Article Title: PHD3-VHL axis controls HIV-2 infection through oxygen-dependent hydroxylation and degradation of Vpx
    Article Snippet: HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit, Cell Signaling) were used at 1:5000 dilution. .. In vitro ubiquitination reactions (10 μL) contained 1 μg recombinant HIV-2 GH-1 Vpx or P41A mutant (synthesized using wheat germ cell-free protein synthesis system), 100 nM ubiquitin-activating enzyme E1 (Merck, #SRP6147), 1 μM UbcH5a (R&D Systems, #E2-616), 100 nM EloB/EloC/VHL/CUL2/RBX1 (R&D Systems, #E3-655–025), 5 μM ubiquitin (Merck, #U5507), 2.5 mM MgATP solution (R&D Systems, #B-20), and 1x E3 Ligase Reaction buffer (R&D Systems, #B-71). .. For PHD3-dependent ubiquitination assays, Vpx was pre-incubated with 300 nM recombinant PHD3/EGLN3 protein (Active Motif, #81033) in PHD3 reaction buffer (20 mM Tris-HCl pH 7.5, 5 mM KCl, 1.5 mM MgCl 2 , 1 mM DTT, 100 μM 2-oxoglutarate, 100 μM ascorbate, and 50 μM (NH 4 ) 2 Fe(SO 4 ) 2 ·6H 2 O) at 30°C for 60 min.

    In Vitro:

    Article Title: PHD3-VHL axis controls HIV-2 infection through oxygen-dependent hydroxylation and degradation of Vpx.
    Article Snippet: HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit, Cell Signaling) were used at 1:5000 dilution. .. In vitro ubiquitination reactions (10 μL) contained 1 μg recombinant HIV-2 GH-1 Vpx or P41A mutant (synthesized using wheat germ cell-free protein synthesis system), 100 nM ubiquitin-activating enzyme E1 (Merck, #SRP6147), 1 μM UbcH5a (R&D Systems, #E2-616), 100 nM EloB/EloC/VHL/CUL2/RBX1 (R&D Systems, #E3-655–025), 5 μM ubiquitin (Merck, #U5507), 2.5 mM MgATP solution (R&D Systems, #B-20), and 1x E3 Ligase Reaction buffer (R&D Systems, #B-71). .. For PHD3-dependent ubiquitination assays, Vpx was pre-incubated with 300 nM recombinant PHD3/EGLN3 protein (Active Motif, #81033) in PHD3 reaction buffer (20 mM Tris-HCl pH 7.5, 5 mM KCl, 1.5 mM MgCl 2 , 1 mM DTT, 100 μM 2-oxoglutarate, 100 μM ascorbate, and 50 μM (NH 4 ) 2 Fe(SO 4 ) 2 ·6H 2 O) at 30°C for 60 min.

    Article Title: PHD3-VHL axis controls HIV-2 infection through oxygen-dependent hydroxylation and degradation of Vpx
    Article Snippet: HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit, Cell Signaling) were used at 1:5000 dilution. .. In vitro ubiquitination reactions (10 μL) contained 1 μg recombinant HIV-2 GH-1 Vpx or P41A mutant (synthesized using wheat germ cell-free protein synthesis system), 100 nM ubiquitin-activating enzyme E1 (Merck, #SRP6147), 1 μM UbcH5a (R&D Systems, #E2-616), 100 nM EloB/EloC/VHL/CUL2/RBX1 (R&D Systems, #E3-655–025), 5 μM ubiquitin (Merck, #U5507), 2.5 mM MgATP solution (R&D Systems, #B-20), and 1x E3 Ligase Reaction buffer (R&D Systems, #B-71). .. For PHD3-dependent ubiquitination assays, Vpx was pre-incubated with 300 nM recombinant PHD3/EGLN3 protein (Active Motif, #81033) in PHD3 reaction buffer (20 mM Tris-HCl pH 7.5, 5 mM KCl, 1.5 mM MgCl 2 , 1 mM DTT, 100 μM 2-oxoglutarate, 100 μM ascorbate, and 50 μM (NH 4 ) 2 Fe(SO 4 ) 2 ·6H 2 O) at 30°C for 60 min.

    Mutagenesis:

    Article Title: PHD3-VHL axis controls HIV-2 infection through oxygen-dependent hydroxylation and degradation of Vpx.
    Article Snippet: HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit, Cell Signaling) were used at 1:5000 dilution. .. In vitro ubiquitination reactions (10 μL) contained 1 μg recombinant HIV-2 GH-1 Vpx or P41A mutant (synthesized using wheat germ cell-free protein synthesis system), 100 nM ubiquitin-activating enzyme E1 (Merck, #SRP6147), 1 μM UbcH5a (R&D Systems, #E2-616), 100 nM EloB/EloC/VHL/CUL2/RBX1 (R&D Systems, #E3-655–025), 5 μM ubiquitin (Merck, #U5507), 2.5 mM MgATP solution (R&D Systems, #B-20), and 1x E3 Ligase Reaction buffer (R&D Systems, #B-71). .. For PHD3-dependent ubiquitination assays, Vpx was pre-incubated with 300 nM recombinant PHD3/EGLN3 protein (Active Motif, #81033) in PHD3 reaction buffer (20 mM Tris-HCl pH 7.5, 5 mM KCl, 1.5 mM MgCl 2 , 1 mM DTT, 100 μM 2-oxoglutarate, 100 μM ascorbate, and 50 μM (NH 4 ) 2 Fe(SO 4 ) 2 ·6H 2 O) at 30°C for 60 min.

    Article Title: PHD3-VHL axis controls HIV-2 infection through oxygen-dependent hydroxylation and degradation of Vpx
    Article Snippet: HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit, Cell Signaling) were used at 1:5000 dilution. .. In vitro ubiquitination reactions (10 μL) contained 1 μg recombinant HIV-2 GH-1 Vpx or P41A mutant (synthesized using wheat germ cell-free protein synthesis system), 100 nM ubiquitin-activating enzyme E1 (Merck, #SRP6147), 1 μM UbcH5a (R&D Systems, #E2-616), 100 nM EloB/EloC/VHL/CUL2/RBX1 (R&D Systems, #E3-655–025), 5 μM ubiquitin (Merck, #U5507), 2.5 mM MgATP solution (R&D Systems, #B-20), and 1x E3 Ligase Reaction buffer (R&D Systems, #B-71). .. For PHD3-dependent ubiquitination assays, Vpx was pre-incubated with 300 nM recombinant PHD3/EGLN3 protein (Active Motif, #81033) in PHD3 reaction buffer (20 mM Tris-HCl pH 7.5, 5 mM KCl, 1.5 mM MgCl 2 , 1 mM DTT, 100 μM 2-oxoglutarate, 100 μM ascorbate, and 50 μM (NH 4 ) 2 Fe(SO 4 ) 2 ·6H 2 O) at 30°C for 60 min.

    Synthesized:

    Article Title: PHD3-VHL axis controls HIV-2 infection through oxygen-dependent hydroxylation and degradation of Vpx.
    Article Snippet: HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit, Cell Signaling) were used at 1:5000 dilution. .. In vitro ubiquitination reactions (10 μL) contained 1 μg recombinant HIV-2 GH-1 Vpx or P41A mutant (synthesized using wheat germ cell-free protein synthesis system), 100 nM ubiquitin-activating enzyme E1 (Merck, #SRP6147), 1 μM UbcH5a (R&D Systems, #E2-616), 100 nM EloB/EloC/VHL/CUL2/RBX1 (R&D Systems, #E3-655–025), 5 μM ubiquitin (Merck, #U5507), 2.5 mM MgATP solution (R&D Systems, #B-20), and 1x E3 Ligase Reaction buffer (R&D Systems, #B-71). .. For PHD3-dependent ubiquitination assays, Vpx was pre-incubated with 300 nM recombinant PHD3/EGLN3 protein (Active Motif, #81033) in PHD3 reaction buffer (20 mM Tris-HCl pH 7.5, 5 mM KCl, 1.5 mM MgCl 2 , 1 mM DTT, 100 μM 2-oxoglutarate, 100 μM ascorbate, and 50 μM (NH 4 ) 2 Fe(SO 4 ) 2 ·6H 2 O) at 30°C for 60 min.

    Article Title: PHD3-VHL axis controls HIV-2 infection through oxygen-dependent hydroxylation and degradation of Vpx
    Article Snippet: HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit, Cell Signaling) were used at 1:5000 dilution. .. In vitro ubiquitination reactions (10 μL) contained 1 μg recombinant HIV-2 GH-1 Vpx or P41A mutant (synthesized using wheat germ cell-free protein synthesis system), 100 nM ubiquitin-activating enzyme E1 (Merck, #SRP6147), 1 μM UbcH5a (R&D Systems, #E2-616), 100 nM EloB/EloC/VHL/CUL2/RBX1 (R&D Systems, #E3-655–025), 5 μM ubiquitin (Merck, #U5507), 2.5 mM MgATP solution (R&D Systems, #B-20), and 1x E3 Ligase Reaction buffer (R&D Systems, #B-71). .. For PHD3-dependent ubiquitination assays, Vpx was pre-incubated with 300 nM recombinant PHD3/EGLN3 protein (Active Motif, #81033) in PHD3 reaction buffer (20 mM Tris-HCl pH 7.5, 5 mM KCl, 1.5 mM MgCl 2 , 1 mM DTT, 100 μM 2-oxoglutarate, 100 μM ascorbate, and 50 μM (NH 4 ) 2 Fe(SO 4 ) 2 ·6H 2 O) at 30°C for 60 min.



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    a Schematic illustration of the catalytic mechanisms employed by major E3 ligase families: RING, HECT, and RBR. Acceptor lysine (on E3 or on substrate) is shown in orange. b Schematic showing the experimental workflow of the E2 discharge assay using free amino acids. c E2 discharge assay using UbcH8∼ISG15 in the presence of various amino acids (50 mM) d E2 discharge assay using UbcH8∼ISG15 in the presence of increasing concentrations of L-lys as indicated. e E2 discharge assay using UbcH7∼Ub in the presence of various amino acids (50 mM f E2 discharge assay using <t>UbcH5A∼Ub</t> in the presence of various amino acids (50 mM)
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    a Schematic illustration of the catalytic mechanisms employed by major E3 ligase families: RING, HECT, and RBR. Acceptor lysine (on E3 or on substrate) is shown in orange. b Schematic showing the experimental workflow of the E2 discharge assay using free amino acids. c E2 discharge assay using UbcH8∼ISG15 in the presence of various amino acids (50 mM) d E2 discharge assay using UbcH8∼ISG15 in the presence of increasing concentrations of L-lys as indicated. e E2 discharge assay using UbcH7∼Ub in the presence of various amino acids (50 mM f E2 discharge assay using <t>UbcH5A∼Ub</t> in the presence of various amino acids (50 mM)
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    Image Search Results


    a Schematic illustration of the catalytic mechanisms employed by major E3 ligase families: RING, HECT, and RBR. Acceptor lysine (on E3 or on substrate) is shown in orange. b Schematic showing the experimental workflow of the E2 discharge assay using free amino acids. c E2 discharge assay using UbcH8∼ISG15 in the presence of various amino acids (50 mM) d E2 discharge assay using UbcH8∼ISG15 in the presence of increasing concentrations of L-lys as indicated. e E2 discharge assay using UbcH7∼Ub in the presence of various amino acids (50 mM f E2 discharge assay using UbcH5A∼Ub in the presence of various amino acids (50 mM)

    Journal: bioRxiv

    Article Title: ISGylation Mechanism Uncovers Conformational Specificity for HECT-family E3 ligase

    doi: 10.1101/2025.09.26.678755

    Figure Lengend Snippet: a Schematic illustration of the catalytic mechanisms employed by major E3 ligase families: RING, HECT, and RBR. Acceptor lysine (on E3 or on substrate) is shown in orange. b Schematic showing the experimental workflow of the E2 discharge assay using free amino acids. c E2 discharge assay using UbcH8∼ISG15 in the presence of various amino acids (50 mM) d E2 discharge assay using UbcH8∼ISG15 in the presence of increasing concentrations of L-lys as indicated. e E2 discharge assay using UbcH7∼Ub in the presence of various amino acids (50 mM f E2 discharge assay using UbcH5A∼Ub in the presence of various amino acids (50 mM)

    Article Snippet: The following plasmids were obtained from Addgene: UBE1 (Addgene plasmid #34965; gift from Cynthia Wolberger), UbcH5A (Addgene plasmid #15782; gift from Wade Harper), and NEDD4L (Addgene plasmid #165103; gift from Arthur Haas).

    Techniques: