Journal: The Journal of Biological Chemistry

Article Title: UBE2O, a host ubiquitin-conjugating enzyme, is a key regulator of hepatitis B virus maturation and egress

doi: 10.1016/j.jbc.2025.110750

Figure Lengend Snippet: HBc/capsid interacts with UBE2O. A , proximity ligation assay (PLA) using a mouse antibody against the HBV capsid (Hyb-3120) and a rabbit antibody against UBE2O in HBV-infected HepG2-NTCP cells at day 6 post-infection. Nuclei were counterstained with DAPI. Bar, = 20 μm. B , PLA using a mouse antibody against the HBV capsid (Hyb-3120) and a rabbit antibody against UBE2O in HepAD38 cells. Tet+, tetracycline-treated cells (low HBV production) and Tet-, tetracycline-untreated cells (high HBV production). Nuclei were counterstained with DAPI. Bar, = 20 μm. C , HepG2-NTCP cells were transfected with Flag-Myc-tagged UBE2O and 2 days post-transfection infected with HBV, as indicated. Six days post-infection, protein lysates were immunoprecipitated (IP) using anti-HBc or anti-capsid (Hyb-3120) antibodies and analyzed by Western blotting (WB) with anti-Flag and anti-HBc antibodies. The expression levels of UBE2O and HBc are shown for comparison (input). D , HepG2-NTCP cells were infected with HBV, as indicated. Six days post-infection, protein lysates were immunoprecipitated (IP) with anti-UBE2O antibodies (lanes one and 2) or an isotype control antibody (lane 3) and analyzed by Western blotting (WB) with anti-HBc and anti-UBE2O antibodies. The expression levels of endogenous UBE2O, HBc and β-actin are shown for comparison (input). E , UBE2O interacts with both capsid assembly-competent (wt, S164A/S172A, and Y132F) and capsid assembly-defective (Y132A, Y132A/S164A/S172A, and W102A) HBc variants. HepG2-NTCP cells were co-transfected with Flag-Myc-tagged UBE2O and wt-HBc or its mutant variants, as indicated. Two days post-transfection, protein lysates were immunoprecipitated (IP) with anti-HBc or anti-Flag antibodies and analyzed by Western blotting (WB) with anti-Flag or anti-HBc antibodies, respectively. The expression levels of UBE2O and HBc are shown for comparison (input). F , the PLA assay was performed using a mouse antibody against the HBV capsid (Hyb-3120) and a rabbit antibody against hypo-pHBc in HBV-infected HepG2-NTCP cells at day 6 post-infection. Nuclei were counterstained with DAPI. Bars, = 20 μm.

Article Snippet: The primary antibodies and reagents used for the Western blotting and immunoprecipitations included antibodies against UBE2O (Novus Biologicals), ubiquitin-HRP (BioLegend), HBV surface antigen (S14, Abcam), β-actin (Abcam), Flag (Sigma-Aldrich), HA (Sigma-Aldrich), α-tubulin (Sigma-Aldrich, USA), lamin A/C (Santa Cruz Biotechnologies), and anti-HA magnetic beads (ThermoFisher Scientific, USA).

Techniques: Proximity Ligation Assay, Infection, Transfection, Immunoprecipitation, Western Blot, Expressing, Comparison, Control, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: UBE2O, a host ubiquitin-conjugating enzyme, is a key regulator of hepatitis B virus maturation and egress

doi: 10.1016/j.jbc.2025.110750

Figure Lengend Snippet: Inhibition of UBE2O expression reduces HBV replication and secretion of HBe antigen in infected HepG2-NTCP cells . A , experimental outline: HepG2-NTCP cells or primary human hepatocytes (PHH; in ) were transfected with control (si_ctrl-A, si_ctrl-B) or UBE2O-specific (si_UBE2O-A, si_UBE2O-B and si_UBE2O-C) siRNAs. Two days after transfection, the cells were infected with the HBV. Six days post-infection, the cells and media were harvested to quantify the expression levels of UBE2O (in B ), intracellular total HBV DNA (in C ), cccDNA (in D ), pgRNA (in E ), and the extracellular HBe antigen (in F ). Data are presented as the mean ± SD from three independent experiments. Color-coded data points distinguish individual replicates from each experiment, and colored horizontal lines indicate the mean values. Statistical significance of differences between UBE2O silencing and control (si_ctrl-A) groups was evaluated by 2-way ANOVA ( p ≥ 0.05 – not significant (ns); ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001).

Article Snippet: The primary antibodies and reagents used for the Western blotting and immunoprecipitations included antibodies against UBE2O (Novus Biologicals), ubiquitin-HRP (BioLegend), HBV surface antigen (S14, Abcam), β-actin (Abcam), Flag (Sigma-Aldrich), HA (Sigma-Aldrich), α-tubulin (Sigma-Aldrich, USA), lamin A/C (Santa Cruz Biotechnologies), and anti-HA magnetic beads (ThermoFisher Scientific, USA).

Techniques: Inhibition, Expressing, Infection, Transfection, Control

Journal: The Journal of Biological Chemistry

Article Title: UBE2O, a host ubiquitin-conjugating enzyme, is a key regulator of hepatitis B virus maturation and egress

doi: 10.1016/j.jbc.2025.110750

Figure Lengend Snippet: UBE2O knockdown in HBV-infected HepG2-NTCP cells led to the inhibition of intracellular nucleocapsid assembly and the secretion of enveloped virions . HepG2-NTCP cells were transfected with two control and three UBE2O-specific siRNAs. Two days post-transfection, the cells were infected with the HBV according to the experimental outline shown in A . Six days post-infection, the cells and media were harvested for analyses of nucleocapsid assembly and virion secretion. A , the protein extracts isolated from HBV-infected HepG2-NTCP cells were analyzed by Western blotting (WB; input) and subjected to immunoprecipitation with anti-capsid (Hyb-3120) antibodies (IP: capsid, WB: HBc). B , downregulation of UBE2O expression in infected HepG2-NTCP cells resulted in decreased levels of intracellular DNA-containing nucleocapsids. Nucleocapsids were immunoprecipitated from protein lysates using anti-capsid antibodies and quantified via qPCR. C , Downregulation of UBE2O expression in infected HepG2-NTCP cells resulted in decreased levels of secreted enveloped virions. Viral particles were immunoprecipitated from the cell culture supernatants using anti-S antibodies and quantified via qPCR. D , the secretion of naked nucleocapsids was not affected by UBE2O downregulation. Naked nucleocapsids were immunoprecipitated from the cell culture supernatants using anti-capsid antibodies, followed by quantification via qPCR. In (B-D), data are presented as the mean ± SD from three independent experiments. Color-coded data points distinguish individual replicates from each experiment, and colored horizontal lines indicate the mean values. The statistical significance of differences between UBE2O silencing and control (si_ctrl-A) groups was evaluated by 2-way ANOVA ( p ≥ 0.05 – not significant (ns); ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001).

Article Snippet: The primary antibodies and reagents used for the Western blotting and immunoprecipitations included antibodies against UBE2O (Novus Biologicals), ubiquitin-HRP (BioLegend), HBV surface antigen (S14, Abcam), β-actin (Abcam), Flag (Sigma-Aldrich), HA (Sigma-Aldrich), α-tubulin (Sigma-Aldrich, USA), lamin A/C (Santa Cruz Biotechnologies), and anti-HA magnetic beads (ThermoFisher Scientific, USA).

Techniques: Knockdown, Infection, Inhibition, Transfection, Control, Isolation, Western Blot, Immunoprecipitation, Expressing, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: UBE2O, a host ubiquitin-conjugating enzyme, is a key regulator of hepatitis B virus maturation and egress

doi: 10.1016/j.jbc.2025.110750

Figure Lengend Snippet: UBE2O and viral capsids associate with MVBs in HBV-infected HepG2-NTCP cells. A–F , HepG2-NTCP cells were either infected with HBV or maintained as uninfected controls. Six days post-infection, cells were immunostained for confocal microscopy analysis (in A–E ) or subjected to PLA (in F ). A , HBV-infected HepG2-NTCP cells were immunostained with anti-capsid ( red ) and anti-UBE2O ( green ) antibodies. White squares indicate enlarged areas of UBE2O and HBV capsid co-localization shown on the right . Nuclei were counterstained with DAPI. Bar, = 10 μm. B , colocalization of HBV capsids ( red ) with CD63 or TSG101 (both green ) in HBV-infected HepG2-NTCP cells. DAPI was used for nuclear staining. Bars, = 10 μm. C , HBV-infected or ( D ) uninfected HepG2-NTCP cells were stained with antibodies against UBE2O ( green ) and CD63 ( red ). White squares indicate enlarged regions of UBE2O and CD63 co-localization, as shown on the right . Nuclei were counterstained with DAPI. Bars, = 10 μm. E , confocal section of HBV-infected HepG2-NTCP cells. Ubiquitin ( red ) and UBE2O ( green ) were stained with specific antibodies, and nuclei were stained with DAPI. White squares indicate enlarged areas of UBE2O and ubiquitinated proteins co-localization, shown on the right . Bars, = 10 μm. F , PLA of the interaction between UBE2O and CD63 in HBV-infected HepG2-NTCP cells was measured 6 days post-infection using Duolink PLA reagents. A mouse isotype antibody was used as a control. Nuclei were counterstained with DAPI. Bars, = 20 μm. G , HepG2-NTCP cells were transfected with wt-HBc, Flag-Myc-tagged wt-UBE2O, Flag-tagged SNF8/EAP30, or Flag-tagged HGS, as indicated. Isolated protein lysates were subjected to immunoprecipitation with anti-capsid antibodies followed by Western blotting with anti-Flag antibodies to detect HGS, SNF8 and UBE2O. The expression levels of transfected proteins were analyzed by Western blotting ( bottom panels ; WB: Flag and WB: HBc). ∗, IgG heavy chain.

Article Snippet: The primary antibodies and reagents used for the Western blotting and immunoprecipitations included antibodies against UBE2O (Novus Biologicals), ubiquitin-HRP (BioLegend), HBV surface antigen (S14, Abcam), β-actin (Abcam), Flag (Sigma-Aldrich), HA (Sigma-Aldrich), α-tubulin (Sigma-Aldrich, USA), lamin A/C (Santa Cruz Biotechnologies), and anti-HA magnetic beads (ThermoFisher Scientific, USA).

Techniques: Infection, Confocal Microscopy, Staining, Ubiquitin Proteomics, Control, Transfection, Isolation, Immunoprecipitation, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: UBE2O, a host ubiquitin-conjugating enzyme, is a key regulator of hepatitis B virus maturation and egress

doi: 10.1016/j.jbc.2025.110750

Figure Lengend Snippet: UBE2O mediates the monoubiquitination of hypophosphorylated HBc localized in the cytoplasm . A , HepG2-NTCP cells were transfected with HA-tagged ubiquitin (HA-Ub) together with Flag-Myc-tagged wt-UBE2O and various wt-HBc expression plasmids that were either untagged (HBc) or tagged at the N-terminus (His-Flag-HBc) or the C-terminus (HBc-Flag). Forty-eight hours after transfection, the cells were fractionated into nuclear ( left ) and cytoplasmic ( right ) extracts. The protein lysates were denatured in 2% SDS and analyzed by immunoprecipitation (IP: anti-HA) followed by Western blotting (WB: hyper-pHBc, HBc and hypo-pHBc). The expression levels of HBc, UBE2O, ubiquitin, lamin A/C (nuclear marker), α-tubulin (cytoplasmic marker) and β-actin (loading control) were analyzed in the input samples. B , the catalytically-defective mutant UBE2O-CD (UBE2O-C1040S) lost the ability to ubiquitinate the HBc/capsids. HepG2-NTCP cells were transfected with HA-tagged ubiquitin, wt-HBc and Flag-Myc-tagged UBE2O-wt or UBE2O-CD. The whole cell lysates were isolated, denatured and analyzed as in ( A ). C , in vitro ubiquitination of wt-HBc with 400 nM UBE2O, 75 nM E1 enzyme, 5 μM ubiquitin-wt (Ub-WT) or methylated ubiquitin (Methyl-Ub). Western blotting with anti-HBc antibodies was used to visualize ubiquitinated HBc, representative of two replicates. D and E , immunoprecipitation and Western blot analysis of cellular lysates from HepG2-NTCP cells transfected with wt-HBc, Flag-Myc-tagged wt-UBE2O and HA-tagged single K (in D ) or K-to-R (in E ) ubiquitin mutants. The ubiquitinated proteins were precipitated with anti-HA antibodies (IP: HA) and analyzed by Western blotting with anti-HBc or anti-hypo-pHBc.

Article Snippet: The primary antibodies and reagents used for the Western blotting and immunoprecipitations included antibodies against UBE2O (Novus Biologicals), ubiquitin-HRP (BioLegend), HBV surface antigen (S14, Abcam), β-actin (Abcam), Flag (Sigma-Aldrich), HA (Sigma-Aldrich), α-tubulin (Sigma-Aldrich, USA), lamin A/C (Santa Cruz Biotechnologies), and anti-HA magnetic beads (ThermoFisher Scientific, USA).

Techniques: Transfection, Ubiquitin Proteomics, Expressing, Immunoprecipitation, Western Blot, Marker, Control, Mutagenesis, Isolation, In Vitro, Methylation

Journal: The Journal of Biological Chemistry

Article Title: UBE2O, a host ubiquitin-conjugating enzyme, is a key regulator of hepatitis B virus maturation and egress

doi: 10.1016/j.jbc.2025.110750

Figure Lengend Snippet: Inhibition of HBc/capsid phosphorylation at S164 and S172 enhances UBE2O-mediated monoubiquitination of HBc/capsid . A , top –Schematic representation of serine-to-alanine mutations in the CTD of HBc. The sequence of wild-type HBc corresponds to HBV genotype A, subtype adw2. Bottom – Analysis of UBE2O-mediated ubiquitination of single or multiple S-to-A HBc mutants. The images display analysis of denatured whole cell lysates immunoprecipitated with anti-HA antibodies (IP: anti-HA) and analyzed by Western blotting with anti-HBc antibodies. B , analysis of UBE2O-mediated ubiquitination of both capsid assembly-competent (wt, S164A/S172A, and Y132F) and capsid assembly-defective (Y132A, Y132A/S164A/S172A, and W102A) HBc mutants. The images display analyses of denatured whole cell lysates immunoprecipitated with anti-HA antibodies (IP: anti-HA) and analyzed by Western blotting with anti-HBc and anti-hypo-pHBc antibodies. C , downregulation of UBE2O led to reduced HBc ubiquitination. HepG2-NTCP cells were transfected with control and UBE2O-specific siRNAs together with HA-Ub, wt-HBc, or S172A-HBc mutant. Denatured protein lysates were immunoprecipitated with anti-HA (Ub) antibodies and precipitated complexes were analyzed by Western blotting with anti-hypo-pHBc antibodies. The expression levels of UBE2O, HBc and ubiquitin were analyzed in the input samples. D , monoubiquitinated HBc assembles into capsids. HepG2-NTCP cells were transfected with wt-HBc, Flag-Myc-tagged wt-UBE2O and HA-tagged ubiquitin, as indicated. The cytoplasmic lysates were subjected to immunoprecipitation with anti-capsid antibodies (IP: anti-capsid, panels on the left, lanes one–4), followed by Western blotting with anti-HBc, anti-hyper-pHBc and anti-hypo-pHBc. The expression levels of HBc, ubiquitin and UBE2O were analyzed by Western blotting (input, panels on the right , lanes five–8).

Article Snippet: The primary antibodies and reagents used for the Western blotting and immunoprecipitations included antibodies against UBE2O (Novus Biologicals), ubiquitin-HRP (BioLegend), HBV surface antigen (S14, Abcam), β-actin (Abcam), Flag (Sigma-Aldrich), HA (Sigma-Aldrich), α-tubulin (Sigma-Aldrich, USA), lamin A/C (Santa Cruz Biotechnologies), and anti-HA magnetic beads (ThermoFisher Scientific, USA).

Techniques: Inhibition, Phospho-proteomics, Sequencing, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Transfection, Control, Mutagenesis, Expressing

Journal: The Journal of Biological Chemistry

Article Title: UBE2O, a host ubiquitin-conjugating enzyme, is a key regulator of hepatitis B virus maturation and egress

doi: 10.1016/j.jbc.2025.110750

Figure Lengend Snippet: UBE2O stimulates HBc/capsid secretion . A and B , analysis of capsid assembly-competent (wt, S164A/S172A, and Y132F) and capsid assembly-defective (Y132A and Y132A/S164A/S172A) HBc variants’ secretion. HepG2-NTCP cells were co-transfected with UBE2O-wt and HBc variants, as indicated. A , the expression levels of intracellular UBE2O and HBc in protein lysates were determined by Western blotting (WB; input). The ability of HBc variants to assemble into capsids was examined by immunoprecipitation with anti-capsid (Hyb-3120) antibodies (IP: capsid; WB: HBc). B , the levels of secreted HBc were analyzed in the cell culture supernatants of transfected cells by Western blotting with HBc, hyper-pHBc and hypo-pHBc antibodies (Extracellular). Graph – Quantification of secreted HBc levels was performed by densitometric analysis of Western blots probed with anti-HBc antibodies, using ImageQuant TL Array software. The data are presented as means ± SDs of one experiment performed in biological replicates (n = 3) shown by orange points. The statistical significance of differences between the control and UBE2O-wt or UBE2O-CD groups was evaluated by a two-tailed t test (∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001). C and D , effect of UBE2O-wt and catalytically-defective (CD) mutant on the secretion of HBc and Ub-fused HBc. C , HepG2-NTCP cells were co-transfected with UBE2O-wt or -CD together with HBc (WT) and Ub-fused HBc (Ub-WT) variant, as indicated. The isolated cytoplasmic protein extracts (Intracellular) were analyzed by immunoprecipitation with anti-Flag (IP:Flag) or anti-HBc (IP:HBc) antibodies followed by Western blotting with anti-HBc or anti-Flag, respectively. The expression levels of UBE2O, HBc, and β-actin (loading control) were analyzed in the input samples (input). D , the levels of secreted HBc and Ub-HBc were analyzed in the medium of transfected cells by Western blotting with anti-HBc, anti-hyper-pHBc and anti-hypo-pHBc antibodies (Extracellular). Graph–Quantitative analysis of secreted HBc and Ub-HBc determined by ELISA in the cell culture supernatants. The data are presented as means ± SDs of one experiment performed in biological replicates (n = 3) shown by orange points. The statistical significance of differences between the control and UBE2O-wt or UBE2O-CD groups was evaluated by a two-tailed t test ( p ≥ 0.05–not significant (ns); ∗∗ p < 0.01).

Article Snippet: The primary antibodies and reagents used for the Western blotting and immunoprecipitations included antibodies against UBE2O (Novus Biologicals), ubiquitin-HRP (BioLegend), HBV surface antigen (S14, Abcam), β-actin (Abcam), Flag (Sigma-Aldrich), HA (Sigma-Aldrich), α-tubulin (Sigma-Aldrich, USA), lamin A/C (Santa Cruz Biotechnologies), and anti-HA magnetic beads (ThermoFisher Scientific, USA).

Techniques: Transfection, Expressing, Western Blot, Immunoprecipitation, Cell Culture, Software, Control, Two Tailed Test, Mutagenesis, Variant Assay, Isolation, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: UBE2O, a host ubiquitin-conjugating enzyme, is a key regulator of hepatitis B virus maturation and egress

doi: 10.1016/j.jbc.2025.110750

Figure Lengend Snippet: UBE2O mediates HBc/capsid multi-monoubiquitination and secretion. A , UBE2O stimulates HBc and capsid monoubiquitination and multi-monoubiquitination. HepG2-NTCP cells were transfected with Flag-Myc-tagged UBE2O-wt (UBE2O upregulation) together with HA-tagged ubiquitin and various HBc (WT, Y132A, and S164A) or Ub-HBc (Ub-WT, Ub-Y132A, and Ub-S164A) variants. The denatured whole cell lysates were immunoprecipitated with anti-HA antibodies (IP: anti-HA) and analyzed by Western blotting with anti-hypo-pHBc antibodies. The expression levels of UBE2O, HBc, and β-actin (loading control) were analyzed in the input samples (input). B , UBE2O depletion leads to the reduction of HBc and capsid monoubiquitination and multi-monoubiquitination. HepG2-NTCP cells were transfected with UBE2O-specific siRNA (si_UBE2O-B; UBE2O downregulation) together with HA-tagged ubiquitin and various HBc or Ub-HBc variants. The samples were analyzed as in ( A ). C , in vitro ubiquitination of Ub-HBc-wt with 400 nM UBE2O, 75 nM E1 enzyme, 5 μM ubiquitin-wt or methylated ubiquitin, as indicated. Western blotting with anti-HBc antibodies was used to visualize ubiquitinated Ub-HBc, representative of two replicates. D and E , Effect of UBE2O overexpression (in D ) or downregulation (in E ) on the secretion of various HBc and Ub-HBc variants. HepG2-NTCP cells were co-transfected with UBE2O-wt ( D ) or UBE2O-specific siRNA (si_UBE2O-B, (E)) together with various HBc and Ub-HBc variants, as indicated. The expression levels of UBE2O, HBc, and β-actin (loading control) were analyzed in the input samples (intracellular). The ability of HBc variants to assemble into capsids was determined by immunoprecipitation with anti-capsid (Hyb-3120) antibodies (IP: capsid; WB: HBc). The levels of secreted HBc and Ub-HBc were analyzed in the cell culture supernatants of transfected cells by Western blotting with anti-HBc, anti-hyper-pHBc and anti-hypo-pHBc antibodies (extracellular). F , comparison of UBE2O- and Alix-mediated secretion of HBc or Ub-fused HBc variants. HepG2-NTCP cells were co-transfected with Flag-Myc-UBE2O or Flag-Alix expression plasmids together with various HBc and Ub-HBc variants, as indicated. The expression levels of UBE2O, Alix, HBc, and β-actin (loading control) were analyzed in the input samples (intracellular). The levels of secreted HBc and Ub-HBc were analyzed in the cell culture supernatants of transfected cells by Western blotting with anti-HBc, anti-hyper-pHBc and anti-hypo-pHBc antibodies (extracellular). Graph – Quantitative analysis of secreted HBc and Ub-HBc determined by ELISA in the cell supernatants. The data are presented as means ± SDs of one experiment performed in biological replicates (n = 3) shown by orange points. Statistical significance of differences between the control and UBE2O- or Alix-transfected groups was evaluated by a two-tailed t test ( p ≥ 0.05 – not significant (ns); ∗∗ p < 0.01; ∗∗∗ p < 0.001).

Article Snippet: The primary antibodies and reagents used for the Western blotting and immunoprecipitations included antibodies against UBE2O (Novus Biologicals), ubiquitin-HRP (BioLegend), HBV surface antigen (S14, Abcam), β-actin (Abcam), Flag (Sigma-Aldrich), HA (Sigma-Aldrich), α-tubulin (Sigma-Aldrich, USA), lamin A/C (Santa Cruz Biotechnologies), and anti-HA magnetic beads (ThermoFisher Scientific, USA).

Techniques: Transfection, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Expressing, Control, In Vitro, Methylation, Over Expression, Cell Culture, Comparison, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: UBE2O, a host ubiquitin-conjugating enzyme, is a key regulator of hepatitis B virus maturation and egress

doi: 10.1016/j.jbc.2025.110750

Figure Lengend Snippet: Overexpression of UBE2O-WT enhances the secretion of both naked nucleocapsids and enveloped virions in HBV-infected HepG2-NTCP cells . A , experimental design: HepG2-NTCP cells were transfected with either wild-type UBE2O (UBE2O-WT), catalytically-defective mutant (UBE2O-CD), or an empty vector (pcDNA). Two days post-transfection, the cells were infected with HBV. At 6 days post-infection, cells and culture supernatants were harvested to quantify intracellular pregenomic RNA (pgRNA), nucleocapsids, and the release of extracellular naked nucleocapsids and virions. B , protein extracts from HBV-infected HepG2-NTCP cells were subjected to Western blot analysis (input) and immunoprecipitation using anti-capsid antibodies (IP: capsid, WB: HBc). C , the levels of HBV pgRNA transcription were analyzed by RT-qPCR and normalized to the housekeeping gene, HPRT1 . D , Analysis of intracellular DNA-containing nucleocapsids in HBV-infected HepG2-NTCP cells using immunoprecipitation with anti-capsids antibodies followed by qPCR. E , UBE2O-WT upregulation enhanced enveloped virion secretion, while the UBE2O-CD mutant reduced it. Viral particles were immunoprecipitated from the culture supernatants using anti-S antibodies. The associated DNA was then isolated and quantified by qPCR. F , UBE2O-WT upregulation enhanced the secretion of naked nucleocapsids. Nucleocapsids were immunoprecipitated from the culture supernatants using anti-capsid antibodies. The associated DNA was then isolated and quantified by qPCR. In ( C–F ), data are presented as the mean ± SD from two independent experiments. Color-coded data points distinguish individual replicates from each experiment, while colored horizontal lines indicate the mean values. Statistical significance of differences between UBE2O-WT, UBE2O-CD and control (pcDNA) groups was evaluated by 2-way ANOVA ( p ≥ 0.05 – not significant (ns); ∗ p < 0.05; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001).

Article Snippet: The primary antibodies and reagents used for the Western blotting and immunoprecipitations included antibodies against UBE2O (Novus Biologicals), ubiquitin-HRP (BioLegend), HBV surface antigen (S14, Abcam), β-actin (Abcam), Flag (Sigma-Aldrich), HA (Sigma-Aldrich), α-tubulin (Sigma-Aldrich, USA), lamin A/C (Santa Cruz Biotechnologies), and anti-HA magnetic beads (ThermoFisher Scientific, USA).

Techniques: Over Expression, Infection, Transfection, Mutagenesis, Plasmid Preparation, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Isolation, Control

Journal: The Journal of Biological Chemistry

Article Title: UBE2O, a host ubiquitin-conjugating enzyme, is a key regulator of hepatitis B virus maturation and egress

doi: 10.1016/j.jbc.2025.110750

Figure Lengend Snippet: Role of UBE2O in HBc/capsid ubiquitination, maturation and virion egress. A , Model for UBE2O-HBc binding . UBE2O-mediated monoubiquitination of HBc enhances the stability of the UBE2O-HBc complex, thereby promoting the subsequent multi-monoubiquitination of HBc. B , HBV infection results in the secretion of diverse viral progeny species, including: (I) HBeAg and spherical HBsAg; (II) naked capsids (NC, genome-positive and genome-negative); (III) infectious genome-positive HBV virions, empty genome-negative HBV virions, and non-infectious empty envelope particles (subviral particles, SVPs). (I) HBeAg and spherical HBsAg are secreted via the host ER-Golgi constitutive secretory pathway. (II) The egress of naked capsids, regardless of their nucleic acid content, is facilitated by Alix via an ESCRT-independent pathway. Interaction between Alix and the NEDD4 family E3 ubiquitin ligase AIP4 drives the release of HBV naked capsids. (III) Assembly and egress of infectious or empty virions rely on the ESCRT machinery, which facilitates capsid sorting into intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). Host components of the MVB pathway support capsid envelopment and virion budding. Our findings demonstrate that UBE2O, an E2/E3 ubiquitin ligase, plays a critical role in these processes. UBE2O recognizes hypophosphorylated HBc and capsids, catalyzes their mono- and multi-monoubiquitination, and facilitates the efficient release of HBV virions. (IV) Hypophosphorylated unassembled HBc is also secreted, likely utilizing the ER-Golgi constitutive secretory pathway shared with HBeAg. Images were created in BioRender.com .

Article Snippet: The primary antibodies and reagents used for the Western blotting and immunoprecipitations included antibodies against UBE2O (Novus Biologicals), ubiquitin-HRP (BioLegend), HBV surface antigen (S14, Abcam), β-actin (Abcam), Flag (Sigma-Aldrich), HA (Sigma-Aldrich), α-tubulin (Sigma-Aldrich, USA), lamin A/C (Santa Cruz Biotechnologies), and anti-HA magnetic beads (ThermoFisher Scientific, USA).

Techniques: Ubiquitin Proteomics, Binding Assay, Infection

Journal: Cell Death and Differentiation

Article Title: Role of Caspase-10-P13tBID axis in erythropoiesis regulation

doi: 10.1038/s41418-022-01066-0

Figure Lengend Snippet: a Representative flow cytometry histograms of a caspase-10 activity assay performed on living cells at indicated days ( N = 4). b (Left panels) Western blots detection of caspase-10 and caspase-8 performed on sorted live cells (PI/annexin-V negative) at indicated time of the CD36 culture for expression of KIT/GPA: day 3: KIT + GPA low , day 6: KIT +/low GPA int , day 8: KIT low GPA high . (*) Caspase-10 activated forms p29 kDa and p17 kDa. (Right panels) Western blots performed on day 5 of the CD36 culture performed at several EPO concentrations in the presence or not of the Fas-L mimetic monoclonal antibody CH11. *Fully activated caspase-8 (P10 kDa), fully activated caspase-10 (P17 kDa) in addition to p29kDa. ( N = 3). c Representative flow cytometry histograms of a caspase-10 activity assay at indicated days, performed on living erythroid cells cultured from bone marrow CD34 + cells ( N = 3). d A schematic representation of the different BID fragments generated by caspase-8 and -10 cleavage. Cleavage at position D60 exposes a glycine to N-myristoylation (M), generating Myr-P15-tBID. A putative caspase-10 cleavage site at position D38 is indicated. e Representative western blot analysis of BID performed on live, sorted cells (PI/annexin-V-negative) at different time points of the culture for expression of KIT/GPA: day 2: KIT + GPA low , day 6: KIT +/- GPA int , day 8: KIT low GPA high ( N = 6). Cleaved forms P18 and P13 are indicated. f Representative western blot analyses of BID performed day 7 on: (+) live sorted cells (PI/annexin-V-negative) or (TL) total lysates of the same culture, expressing wild-type P22-BID (WT), P22-BID-D60A and P22BID-D75A. (P18-tBID (•), P15-tBID (✦), P13-tBID (✶)). ( N = 4). g Western blot anti-BID and anti-caspase-10 performed on day 7 of sh1CASP10-GFP + and control transduced cells culture. h Semi-tryptic analysis of the mass spectrometry data (using Mascot) extracted from the 18 kDa gel area (Mascot score for the upper spectrum: 54) and the 13 kDa gel area (Mascot score for the lower spectrum: 30).

Article Snippet: To inhibit Fas signaling, both human neutralizing anti-Fas SM1/23 and anti-FasL clone 2C10 mAbs (AbCys) were added at a concentration of 10 μg/mL on day 3 of the CD36 culture in 0.5 U/mL of EPO, and were maintained throughout the culture period.

Techniques: Flow Cytometry, Activity Assay, Western Blot, Expressing, Cell Culture, Generated, Control, Mass Spectrometry

Journal: bioRxiv

Article Title: Intricate structure of the interphase chromocenter revealed by the analysis of a factor involved in species formation

doi: 10.1101/441675

Figure Lengend Snippet: ( A ) – Staining of S2 FLAG-HMR line with mouse anti-FLAG (Sigma), anti-HMR 2C10 and rabbit anti-dCenpA antibodies. ( B ) Quantification of centromeres overlapping with HMR. Experiments were performed in a dCenpA-GFP expressing cell line . In total 25 cells and 122 centromeres identified using an anti GFP antibody were counted. ( C ) Confocal (top panel) and stimulated emission depletion (STED) microscopy (middle panel) images of dCenpA and HMR. The bottom panel shows a zoom in of the centromeric region ( D ) Plot profiles of the highest fluorescence intensities of STED images of the chromocenter from S2 cells using anti dCenpA, anti dCenpC and anti HMR antibodies. Intensity graphs were built in ImageJ and normalized to one of the maximum peaks.

Article Snippet: For immunofluorescence and western blotting, rat anti-HMR 2C10 antibody (Helmholtz Zentrum München, ( )) was used in dilution 1:25 (or 1:5 for high resolution microscopy); rat anti-dCenpA 7A2 (Helmholtz Zentrum München, ( )) 1:100 (or 1:50 for high resolution microscopy); rabbit anti-dCenpA (Immunofluorescence grade, Actif Motif) 1:500 (or 1:250 for high resolution microscopy); mouse anti-HP1a C1A9 ( ) 1:100; anti-APEX2 20H10 (raised in this study, Helmholtz Zentrum München) 1:50; Streptavidin-Alexa555 (Thermo Fisher Scientific) 1:400; rabbit anti-histone H3 (Abcam) 1:3000, mouse anti-FLAG (Sigma M2, 1mg/ml) 1:100.

Techniques: Staining, Expressing, Microscopy, Fluorescence

Journal: bioRxiv

Article Title: Intricate structure of the interphase chromocenter revealed by the analysis of a factor involved in species formation

doi: 10.1101/441675

Figure Lengend Snippet: (A) APEX fusion constructs used for proximity based labeling of the chromocenter. (B) Western blots of whole cell lysates from stable cell lines, expressing HMR AP , HP1a AP or APEX NLS . Antibodies used for Western blots were anti-HMR 2C10, anti-HP1a C1A9, anti-APEX2 20H10 and rabbit anti-H3 (for loading control). (C) Localization of APEX fusions in respective stable cell lines. Antibodies used are the same as for Western blotting in ( B ). For cells with centromere staining (indicated by an asterisk) the inlet shows an approx. 2.3-fold zoom of the nucleus. Shown are the stainings of cells with (induced) or without (uninduced) copper mediated induction of the expression of the corresponding fusion protein.

Article Snippet: For immunofluorescence and western blotting, rat anti-HMR 2C10 antibody (Helmholtz Zentrum München, ( )) was used in dilution 1:25 (or 1:5 for high resolution microscopy); rat anti-dCenpA 7A2 (Helmholtz Zentrum München, ( )) 1:100 (or 1:50 for high resolution microscopy); rabbit anti-dCenpA (Immunofluorescence grade, Actif Motif) 1:500 (or 1:250 for high resolution microscopy); mouse anti-HP1a C1A9 ( ) 1:100; anti-APEX2 20H10 (raised in this study, Helmholtz Zentrum München) 1:50; Streptavidin-Alexa555 (Thermo Fisher Scientific) 1:400; rabbit anti-histone H3 (Abcam) 1:3000, mouse anti-FLAG (Sigma M2, 1mg/ml) 1:100.

Techniques: Construct, Labeling, Western Blot, Stable Transfection, Expressing, Control, Staining