Review

ubc9 (MedChemExpress)

MedChemExpress is a verified supplier

MedChemExpress manufactures this product

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

MedChemExpress ubc9
Ubc9, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ubc9/product/MedChemExpress
Average 93 stars, based on 2 article reviews

ubc9 - by Bioz Stars, 2026-05

93/100 stars

Images

Image Search Results

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a Immunofluorescence images and quantification (n = 20) of SUMO1 (green) and SUMO2/3 (red) conjugation in RAW264.7 cells with or without STM 14028S infection (4 hpi). Scale bar, 10 µm. b Core SUMO cycle enzymes and their corresponding primary genes (blue). c, d Transcriptomic and proteomic analyses of SUMO cycle enzyme expression in RAW264.7 cells infected with STM 14028S vs uninfected controls (4 hpi; n = 3). e UBC9 mRNA expression in RAW264.7 cells during STM 14028S infection (0-6 hpi; n = 3). f Immunoblot analysis and quantification of UBC9 protein levels in STM 14028S-infected RAW264.7 cells (0-6 hpi; n = 3). *P < 0.05; ***P < 0.001; ns, not significant.

Article Snippet: Unique primary antibodies used in this study included UBC9 (CST, #4786), SUMO1 (Proteintech, 67557-1-lg), SUMO2/3 (Proteintech, 67154-1-lg), arginine-GlcNAcylation antibody (Abcam, EPR18251), Myd88 (Proteintech, 67969-1-lg), HSPA8 (Proteintech, 10654-1-AP), PDCD4 (Proteintech, 84162-3-RR).

Techniques: Immunofluorescence, Conjugation Assay, Infection, Expressing, Western Blot

a Schematic of the Y2H screening workflow and pairwise interaction validation strategy. b Representative positive colonies on selective plates; four colonies (blue arrows) correspond to Ube2i (encoding UBC9). c Sequencing analysis of positive clones showing that four independent clones encode Ube2i (orange). d Pairwise interaction assay validating the interaction between SseK1 and UBC9. e Co-IP in HEK293T cells confirming the interaction between SseK1 and UBC9. f In vitro pull-down assay demonstrating direct binding between purified SseK1 and UBC9. g BLI analysis showing the binding kinetics between SseK1 and UBC9 in vitro .

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a Schematic of the Y2H screening workflow and pairwise interaction validation strategy. b Representative positive colonies on selective plates; four colonies (blue arrows) correspond to Ube2i (encoding UBC9). c Sequencing analysis of positive clones showing that four independent clones encode Ube2i (orange). d Pairwise interaction assay validating the interaction between SseK1 and UBC9. e Co-IP in HEK293T cells confirming the interaction between SseK1 and UBC9. f In vitro pull-down assay demonstrating direct binding between purified SseK1 and UBC9. g BLI analysis showing the binding kinetics between SseK1 and UBC9 in vitro .

Techniques: Biomarker Discovery, Sequencing, Clone Assay, Co-Immunoprecipitation Assay, In Vitro, Pull Down Assay, Binding Assay, Purification

a HEK293T cells expressing Flag-UBC9 were infected with STM WT or Δ sseK1 , followed by immunoprecipitation and immunoblot analysis of UBC9 Arg-GlcNAcylation. b In vitro enzymatic assays showing Arg-GlcNAcylation of bacterially expressed 6×His-UBC9, detected by immunoblotting. c MS/MS spectrum of Arg-GlcNAcylated Flag-UBC9 peptides. Corresponding b and y ions are indicated along the peptide sequence above the spectrum. d Immunoblot analysis and quantification (normalized to Coomassie-stained protein, n = 3) of Arg-GlcNAcylation of UBC9 and UBC9 R17A. **P < 0.01.

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a HEK293T cells expressing Flag-UBC9 were infected with STM WT or Δ sseK1 , followed by immunoprecipitation and immunoblot analysis of UBC9 Arg-GlcNAcylation. b In vitro enzymatic assays showing Arg-GlcNAcylation of bacterially expressed 6×His-UBC9, detected by immunoblotting. c MS/MS spectrum of Arg-GlcNAcylated Flag-UBC9 peptides. Corresponding b and y ions are indicated along the peptide sequence above the spectrum. d Immunoblot analysis and quantification (normalized to Coomassie-stained protein, n = 3) of Arg-GlcNAcylation of UBC9 and UBC9 R17A. **P < 0.01.

Techniques: Expressing, Infection, Immunoprecipitation, Western Blot, In Vitro, Tandem Mass Spectroscopy, Sequencing, Staining

a Crystal structure of the UBC9-SUMO1 complex (PDB: 2UYZ). b Predicted structure of the UBC9-SUMO2 complex generated using AlphaFold3. Enlarged views of the UBC9-SUMO1 and UBC9-SUMO2 interfaces are highlighted in black boxes. Residues forming hydrogen bonds with UBC9 R17 are shown in stick representation. c Purification of 6×His-UBC9 from E. coli BL21(DE3) co-expressing pSseK1 and pUBC9, followed by immunoblot analysis of UBC9 Arg-GlcNAcylation. d Purification of SUMO1 and SUMO2 proteins from E. coli BL21(DE3) harboring pSUMO1 or pSUMO2, respectively. M, molecular weight marker. e, f MST analysis of the binding affinities between SUMO1 or SUMO2 and unmodified UBC9 or Arg-GlcNAcylated UBC9.

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a Crystal structure of the UBC9-SUMO1 complex (PDB: 2UYZ). b Predicted structure of the UBC9-SUMO2 complex generated using AlphaFold3. Enlarged views of the UBC9-SUMO1 and UBC9-SUMO2 interfaces are highlighted in black boxes. Residues forming hydrogen bonds with UBC9 R17 are shown in stick representation. c Purification of 6×His-UBC9 from E. coli BL21(DE3) co-expressing pSseK1 and pUBC9, followed by immunoblot analysis of UBC9 Arg-GlcNAcylation. d Purification of SUMO1 and SUMO2 proteins from E. coli BL21(DE3) harboring pSUMO1 or pSUMO2, respectively. M, molecular weight marker. e, f MST analysis of the binding affinities between SUMO1 or SUMO2 and unmodified UBC9 or Arg-GlcNAcylated UBC9.

Techniques: Generated, Purification, Expressing, Western Blot, Molecular Weight, Marker, Binding Assay

a Immunoblot analysis of UBC9 Arg-GlcNAcylation mediated by SseK1, SseK2, or SseK3. b AlphaFold-predicted structures of SseK1 (aa 29-336, red), SseK2 (aa 29-348, yellow), and SseK3 (aa 29-335, green), shown with structural alignment using PyMOL. c AlphaFold-modeled structure of the SseK1-UBC9 complex. The enlarged view of the lid-domain region is boxed in black. Residues in the SseK1 lid domain forming hydrogen bonds with UBC9 are shown in stick representation. d Sequence alignment of SseK1, SseK2, and SseK3 generated using ESPript 3.0. e Immunoblot analysis of UBC9 Arg-GlcNAcylation mediated by SseK1, SseK1 A332_Q336del, SseK3, and SseK3 R332delinsARHVQ. f Immunoblot analysis of SUMO2/3 conjugation in RAW264.7 cells infected with STM Δ sseK1 , STM Δ sseK1 complemented with SseK1, or STM Δ sseK1 complemented with either SseK1 D223_D225delinsAAA or SseK1 A332_Q336del (4 hpi). g Phylogenetic analysis of Salmonella Typhimurium SseK1 homologs. Protein sequences homologous to SseK1 (UniProt accession: A0A0H3NK84) were identified using BLASTP analysis against the UniProtKB reference proteomes and Swiss-Prot databases. Sequences with an E-value < 0.05 were selected for phylogenetic analysis. The phylogenetic tree was constructed and visualized using the Interactive Tree of Life (iTOL) online tool.

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a Immunoblot analysis of UBC9 Arg-GlcNAcylation mediated by SseK1, SseK2, or SseK3. b AlphaFold-predicted structures of SseK1 (aa 29-336, red), SseK2 (aa 29-348, yellow), and SseK3 (aa 29-335, green), shown with structural alignment using PyMOL. c AlphaFold-modeled structure of the SseK1-UBC9 complex. The enlarged view of the lid-domain region is boxed in black. Residues in the SseK1 lid domain forming hydrogen bonds with UBC9 are shown in stick representation. d Sequence alignment of SseK1, SseK2, and SseK3 generated using ESPript 3.0. e Immunoblot analysis of UBC9 Arg-GlcNAcylation mediated by SseK1, SseK1 A332_Q336del, SseK3, and SseK3 R332delinsARHVQ. f Immunoblot analysis of SUMO2/3 conjugation in RAW264.7 cells infected with STM Δ sseK1 , STM Δ sseK1 complemented with SseK1, or STM Δ sseK1 complemented with either SseK1 D223_D225delinsAAA or SseK1 A332_Q336del (4 hpi). g Phylogenetic analysis of Salmonella Typhimurium SseK1 homologs. Protein sequences homologous to SseK1 (UniProt accession: A0A0H3NK84) were identified using BLASTP analysis against the UniProtKB reference proteomes and Swiss-Prot databases. Sequences with an E-value < 0.05 were selected for phylogenetic analysis. The phylogenetic tree was constructed and visualized using the Interactive Tree of Life (iTOL) online tool.

Techniques: Western Blot, Sequencing, Generated, Conjugation Assay, Infection, Construct

Approximately two hours after entering macrophages, Salmonella activates its T3SS-2 to deliver effector proteins, including SseK1, SseK2 and SseK3, into the cytoplasm. Among these effectors, SseK1 uniquely engages UBC9 through its unique lid domain and catalyzes arginine-GlcNAcylation of UBC9 at residue R17. This modification prevents UBC9 from forming the UBC9-SUMO thioester intermediate, thereby blocking SUMO conjugation onto a broad set of antimicrobial substrates and impairing their activity, stability or localization. By blocking this SUMO-dependent arm of the host immune response, Salmonella evades host antimicrobial defenses, thereby promoting its survival and enhancing virulence.

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: Approximately two hours after entering macrophages, Salmonella activates its T3SS-2 to deliver effector proteins, including SseK1, SseK2 and SseK3, into the cytoplasm. Among these effectors, SseK1 uniquely engages UBC9 through its unique lid domain and catalyzes arginine-GlcNAcylation of UBC9 at residue R17. This modification prevents UBC9 from forming the UBC9-SUMO thioester intermediate, thereby blocking SUMO conjugation onto a broad set of antimicrobial substrates and impairing their activity, stability or localization. By blocking this SUMO-dependent arm of the host immune response, Salmonella evades host antimicrobial defenses, thereby promoting its survival and enhancing virulence.

Techniques: Residue, Modification, Blocking Assay, Conjugation Assay, Activity Assay

FOXL2 SUMOylation intricately regulates increased expression in CAFs. ( A ) SUMOylation sites of FOXL2 was analyzed by SUMOplot™ analysis. Shown are the top 7 predicted lysine residues; ( B ) In vitro sumoylation assay was employed to confirm the predicted SUMOylation sites using HA-tagged wild-type (WT), or the K25R, K87R, K114R, K150R, and 4KR (where K25, K87, K114 and K150 were all mutated to R). Shown is a representative blot and densitometry analysis of SUMOylated-FOXL2/Total FOXL2; ( C ) The association between SUMOylation and FOXL2 stability was determined by western blotting in HEK-293T cells using α-HA. Cells were transfected with either HA-FOXL2-WT or HA-FOXL2-K25/87R (double mutant, 2KR). The membrane was stripped and re-probed with GAPDH to confirm equivalent loading. Shown is a representative blot and densitometry analysis of three technical replicates; ( D ) Cells were transfected with HA-FOXL2-WT or, HA-FOXL2-2KR ± His-SUMO1. CHX (100 µg/ml) was added to inhibit translation allowing tracking of FOXL2 stability in the presence and absence of His-SUMO1. Blots were probed with α-HA and re-probed with GAPDH to confirm equivalent loading. Shown is a representative blot and densitometry analysis of three technical replicates; ( E ) DMSO (control), MG132 (proteasome inhibitor) or chloroquine (lysosome inhibitor) were added into HEK-293T cells transfected with HA-FOXL2-WT or HA-FOXL2-2KR, before CHX (100 µg/ml) was added. Blots were probed with α-HA and re-probed with GAPDH to confirm equivalent loading. Shown is a representative blot; ( F ) IP assay was used to test the association between FOXL2 SUMOylation and ubiquitination. Lysates obtained from HEK-293T cells transfected with HA-FOXL2-WT or HA-FOXL2-2KR, along with FLAG-UBC9, were immunoprecipitated using α-HA antibody and then probed with α-FLAG antibody. Shown is a representative blot; *, *** P < 0.05, P < 0.001

Journal: BMC Cancer

Article Title: FOXL2 + cancer-associated fibroblasts enhances epithelial ovarian cancer development via TGFβ/Smad signaling

doi: 10.1186/s12885-025-15364-6

Figure Lengend Snippet: FOXL2 SUMOylation intricately regulates increased expression in CAFs. ( A ) SUMOylation sites of FOXL2 was analyzed by SUMOplot™ analysis. Shown are the top 7 predicted lysine residues; ( B ) In vitro sumoylation assay was employed to confirm the predicted SUMOylation sites using HA-tagged wild-type (WT), or the K25R, K87R, K114R, K150R, and 4KR (where K25, K87, K114 and K150 were all mutated to R). Shown is a representative blot and densitometry analysis of SUMOylated-FOXL2/Total FOXL2; ( C ) The association between SUMOylation and FOXL2 stability was determined by western blotting in HEK-293T cells using α-HA. Cells were transfected with either HA-FOXL2-WT or HA-FOXL2-K25/87R (double mutant, 2KR). The membrane was stripped and re-probed with GAPDH to confirm equivalent loading. Shown is a representative blot and densitometry analysis of three technical replicates; ( D ) Cells were transfected with HA-FOXL2-WT or, HA-FOXL2-2KR ± His-SUMO1. CHX (100 µg/ml) was added to inhibit translation allowing tracking of FOXL2 stability in the presence and absence of His-SUMO1. Blots were probed with α-HA and re-probed with GAPDH to confirm equivalent loading. Shown is a representative blot and densitometry analysis of three technical replicates; ( E ) DMSO (control), MG132 (proteasome inhibitor) or chloroquine (lysosome inhibitor) were added into HEK-293T cells transfected with HA-FOXL2-WT or HA-FOXL2-2KR, before CHX (100 µg/ml) was added. Blots were probed with α-HA and re-probed with GAPDH to confirm equivalent loading. Shown is a representative blot; ( F ) IP assay was used to test the association between FOXL2 SUMOylation and ubiquitination. Lysates obtained from HEK-293T cells transfected with HA-FOXL2-WT or HA-FOXL2-2KR, along with FLAG-UBC9, were immunoprecipitated using α-HA antibody and then probed with α-FLAG antibody. Shown is a representative blot; *, *** P < 0.05, P < 0.001

Article Snippet: The different primary antibodies used were α-SUMO1 (67559-1-Ig, 1:3000), α-UBC9 (10070-1-AP, 1:2000), α-Vimentin (10366-1-AP, 1:5000), α-N-cadherin (22018-1-AP, 1:5000), α-E-cadherin (20874-1-AP, 1:20000) (Proteintech, Wuhan, China); α-HA (A02041, 1:800), α-His (A02050, 1:800), α-Flag (A02010, 1:800) (Abbkine, Wuhan, China); α-FOXL2 (ab246511, 1:1000), α-Snail (ab216347, 1:1000) (Abcam, Cambridge, MA, USA); and, α-Smad2/3 (D7G7, 1:1000), α-p-Smad2 (Ser465/467)/Smad3 (Ser423/425) (D27F4, 1:1000) (CST, Boston, MA, USA).

Techniques: Expressing, In Vitro, Western Blot, Transfection, Mutagenesis, Membrane, Control, Ubiquitin Proteomics, Immunoprecipitation

FOXL2 SUMOylation requires SUMO1 and UBC9/UBE2I. ( A ) Putative interaction of FOXL2 with SUMO1, SUMO2, SUMO3, and SUMO4 was detected by String analysis ( https://cn.string-db.org/ ); ( B ) In vitro sumoylation assay was employed to confirm String analysis’s prediction of SUMO1, HEK-293T cells were transfected with HA-FOXL2-WT, FLAG-UBC9, and His-SUMO1-4. In vitro SUMOylation assay using Ni 2+ -NTA pull-down determined that FOXL2 was mainly modified by SUMO1. Shown is a representative blot; ( C ) In vitro sumoylation assay was employed to determine whether UBC9 is compulsorily required for FOXL2 SUMOylation. HEK-293T cells were transfected with HA-FOXL2-WT and His-SUMO1 ± FLAG-UBC9. In vitro SUMOylation using Ni 2+ -NTA pull-down assay determined that UBC9 is required for FOXL2 SUMOylation. Shown is a representative blot; ( D ) In vitro sumoylation assay was employed to determine whether UBC9 is compulsorily required for FOXL2 SUMOylation in CAFs. CAFs were transduced using either a non-targeting control shRNA or shRNA targeting UBC9 . Transduced cells were transfected with HA-FOXL2-WT and His-SUMO1. In vitro SUMOylation using Ni 2+ -NTA pull-down assay determined that UBC9 is required for FOXL2 SUMOylation. Shown is a representative blot

Journal: BMC Cancer

Article Title: FOXL2 + cancer-associated fibroblasts enhances epithelial ovarian cancer development via TGFβ/Smad signaling

doi: 10.1186/s12885-025-15364-6

Figure Lengend Snippet: FOXL2 SUMOylation requires SUMO1 and UBC9/UBE2I. ( A ) Putative interaction of FOXL2 with SUMO1, SUMO2, SUMO3, and SUMO4 was detected by String analysis ( https://cn.string-db.org/ ); ( B ) In vitro sumoylation assay was employed to confirm String analysis’s prediction of SUMO1, HEK-293T cells were transfected with HA-FOXL2-WT, FLAG-UBC9, and His-SUMO1-4. In vitro SUMOylation assay using Ni 2+ -NTA pull-down determined that FOXL2 was mainly modified by SUMO1. Shown is a representative blot; ( C ) In vitro sumoylation assay was employed to determine whether UBC9 is compulsorily required for FOXL2 SUMOylation. HEK-293T cells were transfected with HA-FOXL2-WT and His-SUMO1 ± FLAG-UBC9. In vitro SUMOylation using Ni 2+ -NTA pull-down assay determined that UBC9 is required for FOXL2 SUMOylation. Shown is a representative blot; ( D ) In vitro sumoylation assay was employed to determine whether UBC9 is compulsorily required for FOXL2 SUMOylation in CAFs. CAFs were transduced using either a non-targeting control shRNA or shRNA targeting UBC9 . Transduced cells were transfected with HA-FOXL2-WT and His-SUMO1. In vitro SUMOylation using Ni 2+ -NTA pull-down assay determined that UBC9 is required for FOXL2 SUMOylation. Shown is a representative blot

Techniques: In Vitro, Transfection, Modification, Pull Down Assay, Control, shRNA

ZNF33B enhanced SUMO conjugation to JEV NS5. ( A ) Immunoblot analysis of JEV NS5 SUMOylation from HEK293T cells co-transfected with UBC9-V5, NS5-FLAG, and pEYFP-SUMO1, pEYFP-SUMO2, or pEYFP-SUMO3. The lysates were subjected to precipitation using anti-FLAG antibodies for the enrichment of SUMOylated proteins, followed by probing with the specified antibodies. ( B–D ) Immunoblot analysis of lysates from HEK293T cells co-transfected with NS5-FLAG and pEYFP-SUMO1, pEYFP-SUMO2, or pEYFP-SUMO3. The expression of NS5 was assessed by measuring the band grayscale with the “ImageJ” software. ( E and F ) Immunoblot analysis of lysates from HEK293T and SK6 cells infected with JEV, followed by DMSO or the SUMOylation inhibitor 2-D08 (0–200 μM) treatment for 6 h. ( G and H ) The viral titration analysis of the supernatant in JEV-infected cells treated with 2-D08 was conducted by plaque assay. The statistical analysis of JEV titer in 2-D08-treated cells. ( I ) Immunoblot analysis of UBC9 in HEK293T cells transfected with ZNF33B-MYC, followed by JEV infection. The expression of ZNF33B was assessed by measuring the band grayscale with the “ImageJ” software. ( J ) Immunoblot analysis of lysates from HEK293T cells co-transfected with NS5-FLAG, pEYFP-SUMO1, and UBC9-V5, followed by treatment with 2-D08 (200 µM). The lysates were subjected to precipitation using anti-HA antibodies for the enrichment of SUMOylated proteins and subsequently probed with specific antibodies to confirm the conjugation of SUMO1 to NS5. ( K–M ) Immunoblot analysis of the effect of ZNF33B and its truncations on JEV NS5 SUMOylation from HEK293T cells co-transfected with pEYFP-SUMO1, UBC9-V5, NS5-FLAG, and ZNF33B-HA, ZNF33B ZFs-HA, or ZNF33B ΔZFs-HA. The lysates were subjected to precipitation using anti-FLAG antibodies for the enrichment of SUMOylated proteins, followed by probing with the specified antibodies. All experiments were conducted in triplicate, and data are represented as mean ± SD. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post hoc test (** P < 0.01 and *** P < 0.001).

Journal: Journal of Virology

Article Title: ZNF33B facilitates Japanese encephalitis virus replication by controlling HSPB1/8-mediated SUMOylation of nonstructural protein 5

doi: 10.1128/jvi.00868-25

Figure Lengend Snippet: ZNF33B enhanced SUMO conjugation to JEV NS5. ( A ) Immunoblot analysis of JEV NS5 SUMOylation from HEK293T cells co-transfected with UBC9-V5, NS5-FLAG, and pEYFP-SUMO1, pEYFP-SUMO2, or pEYFP-SUMO3. The lysates were subjected to precipitation using anti-FLAG antibodies for the enrichment of SUMOylated proteins, followed by probing with the specified antibodies. ( B–D ) Immunoblot analysis of lysates from HEK293T cells co-transfected with NS5-FLAG and pEYFP-SUMO1, pEYFP-SUMO2, or pEYFP-SUMO3. The expression of NS5 was assessed by measuring the band grayscale with the “ImageJ” software. ( E and F ) Immunoblot analysis of lysates from HEK293T and SK6 cells infected with JEV, followed by DMSO or the SUMOylation inhibitor 2-D08 (0–200 μM) treatment for 6 h. ( G and H ) The viral titration analysis of the supernatant in JEV-infected cells treated with 2-D08 was conducted by plaque assay. The statistical analysis of JEV titer in 2-D08-treated cells. ( I ) Immunoblot analysis of UBC9 in HEK293T cells transfected with ZNF33B-MYC, followed by JEV infection. The expression of ZNF33B was assessed by measuring the band grayscale with the “ImageJ” software. ( J ) Immunoblot analysis of lysates from HEK293T cells co-transfected with NS5-FLAG, pEYFP-SUMO1, and UBC9-V5, followed by treatment with 2-D08 (200 µM). The lysates were subjected to precipitation using anti-HA antibodies for the enrichment of SUMOylated proteins and subsequently probed with specific antibodies to confirm the conjugation of SUMO1 to NS5. ( K–M ) Immunoblot analysis of the effect of ZNF33B and its truncations on JEV NS5 SUMOylation from HEK293T cells co-transfected with pEYFP-SUMO1, UBC9-V5, NS5-FLAG, and ZNF33B-HA, ZNF33B ZFs-HA, or ZNF33B ΔZFs-HA. The lysates were subjected to precipitation using anti-FLAG antibodies for the enrichment of SUMOylated proteins, followed by probing with the specified antibodies. All experiments were conducted in triplicate, and data are represented as mean ± SD. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post hoc test (** P < 0.01 and *** P < 0.001).

Article Snippet: The antibody probed by UBC9 (Cat. # HA500401 ) was obtained from HUABIO (China).

Techniques: Conjugation Assay, Western Blot, Transfection, Expressing, Software, Infection, Titration, Plaque Assay

Journal: Journal of Virology

Article Title: ZNF33B facilitates Japanese encephalitis virus replication by controlling HSPB1/8-mediated SUMOylation of nonstructural protein 5

doi: 10.1128/jvi.00868-25

Figure Lengend Snippet: Lysine residues 269 and 846 are critical for ZNF33B-promoted NS5 SUMOylation. ( A ) The diagram illustration of SUMOylation site prediction from Advanced Services of GPS-SUMO 2.0 ( https://sumo.biocuckoo.cn/online.php ), JASSA version 4 ( http://www.jassa.fr ), and SUMOplot Analysis Program ( https://www.abcepta.com/sumoplot ) websites. The overlap of predicted sites is shown in the bottom table. ( B–D ) Immunoblot analysis of NS5 SUMOylation in HEK293T cells co-transfected with UBC9-V5, pEYFP-SUMO1, ZNF33B-MYC, and the WT or mutant constructs of NS5-FLAG, with arginine replacement of the lysine residues at positions 269 ( B ), 287 ( C ), and 846 ( D ). The lysates were subjected to precipitation using anti-GFP antibodies for the enrichment of SUMOylated proteins, followed by probing with the specified antibodies. ( E–G ) Immunoblot analysis of the K269R ( E ), K287R ( F ), and K846R ( G ) mutations on the association of ZNF33B with JEV NS5 by immunoprecipitation of lysates from HEK293T cells transfected with ZNF33B-MYC and NS5-FLAG. The cell lysates were immunoprecipitated with anti-FLAG antibody. ( H ) Confocal microscope observation of the colocalization of ZNF33B with the WT or mutations of NS5 in HEK293T cells transfected with ZNF33B-HA and NS5-FLAG with arginine replacement of the lysine residues at positions 269, 287, and 846. Scale bar, 2 µm. ( I ) Left, sequence alignment of SUMO interaction motifs (SIMs) derived from some E3 SUMO enzymes or flaviviruses NS5 proteins, with the dotted line indicating potential SIM. Right, a diagram depicting the SIMs generated by WebLogo ( https://weblogo.berkeley.edu/logo.cgi ). ( J ) The interaction model of JEV NS5 SIM and SUMO1 was generated by AlphaFold Server. The putative interacting amino acids were identified and annotated using PyMOL software. ( K ) Immunoblot analysis of JEV NS5 SUMOylation in HEK293T cells co-transfected with UBC9-V5, pEYFP-SUMO1, ZNF33B-MYC, and the WT or mutant construct of NS5-FLAG, with arginine replacement of the SIM (V-I-D-L to 4R). The lysates were subjected to precipitation using anti-FLAG antibodies for the enrichment of SUMOylated proteins, followed by probing with the specified antibodies. ( L ) Immunoblot analysis of the SIM mutations on the association of ZNF33B with JEV NS5 by immunoprecipitation of lysates from HEK293T cells transfected with ZNF33B-MYC and NS5-FLAG. The cell lysates were immunoprecipitated with anti-FLAG antibody.

Article Snippet: The antibody probed by UBC9 (Cat. # HA500401 ) was obtained from HUABIO (China).

Techniques: Western Blot, Transfection, Mutagenesis, Construct, Immunoprecipitation, Microscopy, Sequencing, Derivative Assay, Generated, Software

SUMOylation of JEV NS5 hinders its ubiquitination through competitive binding to lysine residues 269 and 846. ( A ) Immunoblot analysis of the effect of JEV NS5 SUMOylation on its ubiquitination in HEK293T cells co-transfected with UBC9-V5, pEYFP-SUMO1, NS5-FLAG, and Ubi-HA. The cell lysates were immunoprecipitated with anti-FLAG antibody. ( B ) Immunoblot analysis of the effect of JEV NS5 deSUMOylation on its ubiquitination in HEK293T cells co-transfected with NS5-FLAG and Ubi-HA, followed by 2-D08 (200 µM) treatment for 6 h. The cell lysates were immunoprecipitated with anti-FLAG antibody. ( C–E ) Immunoblot analysis of the K269R ( C ), K287R ( D ), and K846R ( E ) mutations of JEV NS5 on its ubiquitination by immunoprecipitation of lysates from HEK293T cells transfected with Ubi-HA and the WT or mutant constructs of NS5-FLAG, with arginine replacement of the lysine residues at positions 269 ( C ), 287 ( D ), and 846 ( E ). The cell lysates were immunoprecipitated with anti-FLAG antibody. ( F ) Immunoblot analysis of the SIM mutations of JEV NS5 on its ubiquitination by immunoprecipitation of lysates from HEK293T cells transfected with Ubi-HA and the WT or mutant construct of NS5-FLAG, with arginine replacement of the SIM (V-I-D-L to 4R). The cell lysates were immunoprecipitated with anti-FLAG antibody.

Journal: Journal of Virology

Article Title: ZNF33B facilitates Japanese encephalitis virus replication by controlling HSPB1/8-mediated SUMOylation of nonstructural protein 5

doi: 10.1128/jvi.00868-25

Figure Lengend Snippet: SUMOylation of JEV NS5 hinders its ubiquitination through competitive binding to lysine residues 269 and 846. ( A ) Immunoblot analysis of the effect of JEV NS5 SUMOylation on its ubiquitination in HEK293T cells co-transfected with UBC9-V5, pEYFP-SUMO1, NS5-FLAG, and Ubi-HA. The cell lysates were immunoprecipitated with anti-FLAG antibody. ( B ) Immunoblot analysis of the effect of JEV NS5 deSUMOylation on its ubiquitination in HEK293T cells co-transfected with NS5-FLAG and Ubi-HA, followed by 2-D08 (200 µM) treatment for 6 h. The cell lysates were immunoprecipitated with anti-FLAG antibody. ( C–E ) Immunoblot analysis of the K269R ( C ), K287R ( D ), and K846R ( E ) mutations of JEV NS5 on its ubiquitination by immunoprecipitation of lysates from HEK293T cells transfected with Ubi-HA and the WT or mutant constructs of NS5-FLAG, with arginine replacement of the lysine residues at positions 269 ( C ), 287 ( D ), and 846 ( E ). The cell lysates were immunoprecipitated with anti-FLAG antibody. ( F ) Immunoblot analysis of the SIM mutations of JEV NS5 on its ubiquitination by immunoprecipitation of lysates from HEK293T cells transfected with Ubi-HA and the WT or mutant construct of NS5-FLAG, with arginine replacement of the SIM (V-I-D-L to 4R). The cell lysates were immunoprecipitated with anti-FLAG antibody.

Article Snippet: The antibody probed by UBC9 (Cat. # HA500401 ) was obtained from HUABIO (China).

Techniques: Ubiquitin Proteomics, Binding Assay, Western Blot, Transfection, Immunoprecipitation, Mutagenesis, Construct

ZNF33B recruits HSPB1 to facilitate JEV NS5 SUMOylation. ( A ) Silver staining of JEV NS5-associated proteins immunoprecipitated by anti-HA antibodies probe with cell lysates from SK6 cells. The arrows indicate the additional band presented in JEV NS5-associated proteins. ( B and C ) Differentially expressed proteins analyzed by mass spectrometry. ( D and E ) Immunoblot analysis of the association of NS5 with HSPB1 or HSPB8 by immunoprecipitation of lysates from HEK293T cells transfected with NS5-FLAG and HSPB1-HA or HSPB8-HA. The cell lysates were immunoprecipitated with anti-FLAG antibody. ( F and G ) Confocal microscope observation of the colocalization of NS5 with HSPB1 and HSPB8 in HEK293T cells transfected with NS5-FLAG with HSPB1-HA or HSPB8-HA. Scale bar, 2 µm. ( H and I ) Immunoblot analysis of the expression of JEV NS3 and NS5 in HEK293T cells transfected with HSPB1-HA or HSPB8-HA, followed by JEV infection. ( J and K ) Immunoblot analysis of the effect of HSPB1 and HSPB8 on JEV NS5 SUMOylation from HEK293T cells co-transfected with pEYFP-SUMO1, UBC9-V5, NS5-FLAG, and HSPB1-HA or HSPB8-HA. The lysates were subjected to precipitation using anti-FLAG antibodies for the enrichment of SUMOylated proteins, followed by probing with the specified antibodies. ( L ) Immunoblot analysis of the association of UBC9 with HSPB1 or HSPB8 by immunoprecipitation of lysates from HEK293T cells transfected with UBC9-V5 and HSPB1-HA or HSPB8-HA. The cell lysates were immunoprecipitated with anti-HA antibody. ( M ) Immunoblot analysis of the association of ZNF33B with HSPB1 or HSPB8 by immunoprecipitation of lysates from HEK293T cells transfected with ZNF33B-FLAG and HSPB1-HA or HSPB8-HA. The cell lysates were immunoprecipitated with anti-HA antibody. ( N ) Immunoblot analysis of HSPB1 and HSPB8 in HEK293T cells transfected with ZNF33B-HA, followed by JEV infection. The expression of HSPB1 and HSPB8 was assessed by measuring the band grayscale with the “ImageJ” software. ( O ) Immunoblot analysis of the effect of HSPB1 and HSPB8 depletion on the protein level of ZNF33B-promoted NS5 in HEK293T cells transfected with ZNF33B-MYC, NS5-FLAG, and siRNAs targeting HSPB1 and HSPB8. The expression of NS5 was assessed by measuring the band grayscale with the “ImageJ” software. ( P ) Immunoblot analysis of the effect of HSPB1 and HSPB8 depletion on ZNF33B-promoted JEV NS5 SUMOylation from HEK293T cells co-transfected with ZNF33B-MYC, pEYFP-SUMO1, UBC9-V5, NS5-FLAG, and siRNAs targeting HSPB1 and HSPB8. The lysates were subjected to precipitation using anti-FLAG antibodies for the enrichment of SUMOylated proteins, followed by probing with the specified antibodies.

Journal: Journal of Virology

Article Title: ZNF33B facilitates Japanese encephalitis virus replication by controlling HSPB1/8-mediated SUMOylation of nonstructural protein 5

doi: 10.1128/jvi.00868-25

Figure Lengend Snippet: ZNF33B recruits HSPB1 to facilitate JEV NS5 SUMOylation. ( A ) Silver staining of JEV NS5-associated proteins immunoprecipitated by anti-HA antibodies probe with cell lysates from SK6 cells. The arrows indicate the additional band presented in JEV NS5-associated proteins. ( B and C ) Differentially expressed proteins analyzed by mass spectrometry. ( D and E ) Immunoblot analysis of the association of NS5 with HSPB1 or HSPB8 by immunoprecipitation of lysates from HEK293T cells transfected with NS5-FLAG and HSPB1-HA or HSPB8-HA. The cell lysates were immunoprecipitated with anti-FLAG antibody. ( F and G ) Confocal microscope observation of the colocalization of NS5 with HSPB1 and HSPB8 in HEK293T cells transfected with NS5-FLAG with HSPB1-HA or HSPB8-HA. Scale bar, 2 µm. ( H and I ) Immunoblot analysis of the expression of JEV NS3 and NS5 in HEK293T cells transfected with HSPB1-HA or HSPB8-HA, followed by JEV infection. ( J and K ) Immunoblot analysis of the effect of HSPB1 and HSPB8 on JEV NS5 SUMOylation from HEK293T cells co-transfected with pEYFP-SUMO1, UBC9-V5, NS5-FLAG, and HSPB1-HA or HSPB8-HA. The lysates were subjected to precipitation using anti-FLAG antibodies for the enrichment of SUMOylated proteins, followed by probing with the specified antibodies. ( L ) Immunoblot analysis of the association of UBC9 with HSPB1 or HSPB8 by immunoprecipitation of lysates from HEK293T cells transfected with UBC9-V5 and HSPB1-HA or HSPB8-HA. The cell lysates were immunoprecipitated with anti-HA antibody. ( M ) Immunoblot analysis of the association of ZNF33B with HSPB1 or HSPB8 by immunoprecipitation of lysates from HEK293T cells transfected with ZNF33B-FLAG and HSPB1-HA or HSPB8-HA. The cell lysates were immunoprecipitated with anti-HA antibody. ( N ) Immunoblot analysis of HSPB1 and HSPB8 in HEK293T cells transfected with ZNF33B-HA, followed by JEV infection. The expression of HSPB1 and HSPB8 was assessed by measuring the band grayscale with the “ImageJ” software. ( O ) Immunoblot analysis of the effect of HSPB1 and HSPB8 depletion on the protein level of ZNF33B-promoted NS5 in HEK293T cells transfected with ZNF33B-MYC, NS5-FLAG, and siRNAs targeting HSPB1 and HSPB8. The expression of NS5 was assessed by measuring the band grayscale with the “ImageJ” software. ( P ) Immunoblot analysis of the effect of HSPB1 and HSPB8 depletion on ZNF33B-promoted JEV NS5 SUMOylation from HEK293T cells co-transfected with ZNF33B-MYC, pEYFP-SUMO1, UBC9-V5, NS5-FLAG, and siRNAs targeting HSPB1 and HSPB8. The lysates were subjected to precipitation using anti-FLAG antibodies for the enrichment of SUMOylated proteins, followed by probing with the specified antibodies.

Article Snippet: The antibody probed by UBC9 (Cat. # HA500401 ) was obtained from HUABIO (China).

Techniques: Silver Staining, Immunoprecipitation, Mass Spectrometry, Western Blot, Transfection, Microscopy, Expressing, Infection, Software

Review

Structured Review

Images

Similar Products

Image Search Results