Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2 and either pRK5-HA-Ub or pRK5-HA-Ub-K6 and with either pUC19-HBV-C-AT_JPN, carrying a 1.3-mer overlength HBV, or pUC19-HBV-C-AT_JPN(ΔHBx), carrying a 1.3-mer overlength HBV lacking HBx gene expression. At 48 h posttransfection, cells were harvested and lysed under denaturing conditions. Cell lysates were immunoprecipitated with anti-FLAG M2 affinity beads, followed by immunoblotting with anti-HA PAb (top panel) or anti-FLAG MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-FLAG MAb (fourth panel), anti-HBx PAb (fifth panel), anti-HBc MAb (sixth panel), and anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. (B) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2, pEF1A-HBx-Myc-His 6 , pCAG-HUWE1, and either pRK5-HA-Ub or pRK5-HA-Ub-K6. At 48 h posttransfection, cells were harvested. Cell lysates were subjected to immunoprecipitation with anti-FLAG beads, followed by immunoblotting with anti-HA PAb (top panel) or anti-Nrf2 MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-Nrf2 MAb (fourth panel), anti-HUWE1 PAb (fifth panel), anti-c-Myc MAb (sixth panel), and anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. (C) HepG2 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 24 h post-siRNA transfection, cells were co-transfected with pCAG-FLAG-Nrf2, pRK5-HA-Ub-K6, pEF1A-HBx-Myc-His 6 , and the HUWE1 siRNA-resistant plasmid pCAG-HUWE1_R. At 48 h posttransfection, cells were harvested. Cell lysates were subjected to immunoprecipitation with anti-FLAG beads, followed by immunoblotting with anti-HA PAb (top panel) or anti-FLAG MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-Nrf2 MAb (fourth panel), anti-HUWE1 PAb (fifth panel), anti-c-Myc MAb (sixth panel), or anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. HUWE1_R, siRNA-resistant HUWE1. (D) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2, pRK5-HA-Ub-K6, pEF1A-HBx-Myc-His 6 and either pCAG-HUWE1(WT) or the catalytically inactive mutant pCAG-HUWE1(C4341A). At 48 h posttransfection, cells were harvested. Cell lysates were subjected to immunoprecipitation with anti-FLAG beads, followed by immunoblotting with anti-HA PAb (top panel) or anti-Nrf2 MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-HUWE1 PAb (fourth panel), anti-Nrf2 MAb (fifth panel), anti-c-Myc MAb (sixth panel), or anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. Data in panels A to D are representative of three independent experiments.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Gene Expression, Immunoprecipitation, Western Blot, Control, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with pCAG-HA-HUWE1 and pEF1A-HBx-Myc-His 6 . At 48 h posttransfection, cells were harvested. Cell lysates were immunoprecipitated with anti-HA PAb, followed by immunoblotting with anti-c-Myc MAb (top panel) or anti-HA PAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-c-Myc MAb (fourth panel), and anti-GAPDH MAb (fifth panel). GAPDH served as a loading control. (B) HepG2 cells were co-transfected with pCAG-HA-HUWE1 and either pUC19-HBV-C-AT_JPN or pUC19-HBV-C-AT_JPN (ΔHBx). At 48 h posttransfection, cells were harvested. Cell lysates were immunoprecipitated with anti-HA PAb, followed by immunoblotting with anti-HBx PAb (top panel), anti-HBc MAb (second panel), or anti-HA PAb (third panel). Input samples were immunoblotted with anti-HA PAb (fourth panel), anti-HBx PAb (fifth panel), anti-HBc Mab (sixth panel), and anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. (C) HepG2 cells were transfected with pEF1A-HBx-Myc-His 6 . At 48 h posttransfection, cells were harvested. Cell lysates were immunoprecipitated with anti-HUWE1 PAb or rabbit IgG (control), followed by immunoblotting with anti-c-Myc MAb (top panel) or anti-HUWE1 PAb (second panel). Input samples were immunoblotted with anti-c-Myc MAb (third panel), anti-HUWE1 PAb (fourth panel), and anti-GAPDH MAb (fifth panel). GAPDH served as a loading control. Data are representative of three independent experiments.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A) HepG2 cells were transfected with pCAG-HA-HUWE1 alone, pEF1A-HBx-Myc-His6 alone, or both plasmids. At 48 h posttransfection, cells were fixed and subjected to indirect immunofluorescence staining with anti-HUWE1 rabbit PAb followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), and anti-c-Myc mouse MAb followed by Alexa Fluor 594-conjugated goat anti-mouse IgG (red). Nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 10 μm. (B) HepG2 cells were co-transfected with pCAG-HA-HUWE1 and pEF1A-HBx-Myc-His 6 . At 48 h posttransfection, cells were fixed and subjected to an in situ proximity ligation assay (PLA) using both anti-HA rabbit PAb and anti-c-Myc mouse MAb. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 10 μm. Data in panels A and B are representative of three independent experiments.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Immunofluorescence, Staining, In Situ, Proximity Ligation Assay

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with pCAG-HA-HUWE1, pCAG-FLAG-Nrf2, and pEF1A-HBx-Myc-His 6 . At 48 h posttransfection, cells were harvested. Cell lysates were immunoprecipitated with anti-HA PAb, followed by immunoblotting with anti-Nrf2 MAb (top panel), anti-c-Myc MAb (second panel), and anti-HUWE1 PAb (third panel). Input samples were immunoblotted with the indicated antibodies, and GAPDH served as a loading control. (B) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2 and pCAG-HA-HUWE1 and either pEGFP-C3 (upper panel) or pEGFP-C3-HBx (bottom panel). At 48 h posttransfection, the cells were fixed and incubated with anti-FLAG mouse MAb and anti-HA rabbit PAb, followed by an in situ PLA. Scale bar, 10 μm. (C) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2, pRK5-HA-Ub-K6, pCAG-HUWE1, and pEF1A-HBx-Myc-His 6. At 48 h posttransfection, cells were harvested. Cell lysates were immunoprecipitated with anti-FLAG beads, followed by immunoblotting with anti-HA PAb (top panel) or anti-Nrf2 MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-Nrf2 MAb (fourth panel), anti-HUWE1 PAb (fifth panel), anti-c-Myc MAb (sixth panel), or anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. Data in panels A to C are representative of three independent experiments.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, Incubation, In Situ

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A to D) HepG2 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 24 h post-siRNA transfection, cells were co-transfected with pCAG-FLAG-Nrf2 and either pEF1A-Myc-His 6 (panels A and B) or pEF1A-HBx-Myc-His 6 (panels C and D). At 48 h posttransfection, cells were treated with 100 µg/mL cycloheximide (CHX) for 0, 1, 2, 4, or 6 h prior to harvesting. Cell lysates were then subjected to immunoblotting with the indicated antibodies. GAPDH served as a loading control. (B, D) Specific signals were quantified by densitometry, and the percentage of remaining FLAG-Nrf2 at each time point was compared with that at time zero. Closed triangles (▴) indicate control siRNA, and closed circles (●) indicate HUWE1 siRNA. Data are mean ± standard errors of the mean (SEM) of three independent experiments. * P < 0.05 compared with the control siRNA by Student’s t test. (E) HepG2 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 24 h post-siRNA transfection, cells were further transfected with pCAG-FLAG-Nrf2 and either pEF1A-Myc-His 6 or pEF1A-HBx-Myc-His 6 . At 48 h posttransfection, cells were harvested and subjected to subcellular fractionation. Cytoplasmic and nuclear factions were analyzed by immunoblotting with the indicated antibodies. The relative levels of nuclear Nrf2 were quantified by densitometry and are indicated below each lane. IĸBα and histone H3 served as cytoplasmic and nuclear markers, respectively. The western blots are representative of three independent experiments.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Control, Western Blot, Cell Fractionation

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A to C) HepG2 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 24 h post-siRNA transfection, cells were co-transfected with pUC19-HBV-D-IND60 and the HUWE1 siRNA-resistant plasmid pCAG-HUWE1_R. At 48 h post-plasmid transfection, cells were harvested. Total RNA was extracted, and HBV RNA (A) and pgRNA (B) were quantified by RT-qPCR and normalized to GAPDH mRNA. Data represent the means ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. HUWE1_R, siRNA-resistant HUWE1. (C) Cell lysates were subjected to immunoblotting using anti-HUWE1 PAb (top panel), anti-HBc MAb (middle panel), and anti-GAPDH MAb (bottom panel), with GAPDH as the loading control. The relative levels of HBc were quantified by densitometry and are indicated below each lane. (D-F) Hep38.7-Tet cells were transfected with 40 nM HUWE1 siRNA or control siRNA and subsequently cultured for 7 days without doxycycline to induce HBV replication. Total RNA was extracted, and HBV RNA (D) and pgRNA (E) were quantified by RT-qPCR. Data represent the means ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. (F) Cell lysates were subjected to immunoblotting using anti-HUWE1 PAb (top panel), anti-HBc MAb (middle panel), and anti-GAPDH MAb (bottom panel), with GAPDH as the loading control. The relative levels of HBc were quantified by densitometry and are indicated below each lane. (G) HepG2-NTCP-Myc-His 6 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 48 h posttransfection, cells were infected with recombinant HBV expressing the NanoLuc (NL) reporter gene (HBV/NL) at 100 genome equivalents (GEq)/cell. At 4 days postinfection (dpi), cells were lysed to measure NL activity as an indicator of HBV replication. Results represent the mean ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. (H-J) HepG2-NTCP-Myc-His 6 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 48 h posttransfection, cells were infected with HBV at 1,000 GEq/cell, and harvested at 5 dpi. Total RNA was extracted, and HBV RNA (H) and pgRNA (I) were quantified by qPCR. Data represent the means ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. (J) Cell lysates were subjected to immunoblotting using anti-HUWE1 PAb (top panel), anti-HBc MAb (middle panel), and anti-GAPDH MAb (bottom panel), with GAPDH as the loading control. The relative levels of HBc were quantified by densitometry and are indicated below each lane. (K-L) PXB cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 48 h posttransfection, cells were infected with HBV at 1,000 GEq/cell and harvested at 5 dpi. Total RNA was extracted, and HUWE1 mRNA (K) was quantified by RT-qPCR. Data represent the means ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. (L) Cell lysates were subjected to immunoblotting using anti-HUWE1 PAb (top panel), anti-HBc MAb (middle panel), and anti-GAPDH MAb (bottom panel), with GAPDH as the loading control. The relative levels of HBc were quantified by densitometry and are indicated below each lane.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Cell Culture, Infection, Recombinant, Expressing, Activity Assay

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2, pRK5-HA-Ub-K6, and pEF1A-HBx-Myc-His 6 . At 24 h posttransfection, cells were treated with 10 µM BI8626 or equivalent volume of dimethyl sulfoxide (DMSO; vehicle control) for 48 h prior to harvesting. Cell lysates were immunoprecipitated with anti-FLAG beads and subjected to immunoblotting with anti-HA PAb (top panel) or anti-Nrf2 MAb (second panel). Input samples were immunoblotted with the indicated antibodies. GAPDH served as a loading control. (B) Cell viability of HepG2 cells treated with 10 µM BI8626 for 48 h, assessed by CellTiter-Glo v2.0 ATP assay. Results represent the mean values ± SEM from three biological replicates. (C to E) HepG2-NTCP-Myc-His 6 cells were infected with HBV at 1,000 GEq/cell. At 24 h postinfection, cells were treated with 50 µM BI8626 and maintained until harvesting. At 10 dpi, total RNA was extracted, and HBV RNA (C) and pgRNA (D) were quantified by RT-qPCR. Data represent the means ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. (E) Cell lysates were analyzed by immunoblotting with anti-HUWE1 PAb (top panel), anti-HBc MAb (middle panel), and anti-GAPDH MAb (bottom panel), with GAPDH serving as a loading control. The relative levels of HBc were quantified by densitometry and are indicated below each lane. (F) Cell viability of HepG2-NTCP-Myc-His 6 cells treated with 50 µM BI8626 for 48 h, assessed by CellTiter-Glo 2.0. Data represent the means ± SEM from three biological replicates.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Control, Immunoprecipitation, Western Blot, ATP Assay, Infection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: Under normal conditions (upper panel), HUWE1 does not associate with Nrf2. Keap1 bridges Nrf2 to the Cullin3 E3 ligase complex, leading to K48-linked polyubiquitylation and proteasomal degradation of Nrf2. Upon HBV infection (lower panel), HBx recruits HUWE1 to form a ternary HUWE1/HBx/Nrf2 complex in the cytoplasm. HUWE1 then adds K6-linked polyubiquitylation chains to Nrf2, switching the ubiquitin code from K48 to K6 linkage. K6-linked polyubiquitylation stabilizes Nrf2, promotes its nuclear translocation, and leads transcriptional activation of antioxidant response element (ARE)-driven genes. In the nucleus, Nrf2 suppresses HBV core, PreS1, and PreS2/S promoter activities, thereby suppressing HBV replication. Meanwhile, Nrf2 also suppresses HBV replication through its target gene Prdx1, which facilitates HBV RNA degradation, as we reported previously .

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Infection, Ubiquitin Proteomics, Translocation Assay, Activation Assay

Journal: Cancer Medicine

Article Title: Genomic characterization of individuals presenting extreme phenotypes of high and low risk to develop tobacco‐induced lung cancer

doi: 10.1002/cam4.1500

Figure Lengend Snippet: Univariate and multivariate Cox regression analyses of PDE10A protein expression for recurrence‐free survival (RFS) and overall survival (OS) in patients with stage I–II NSCLC

Article Snippet: We used an anti‐human PDE10A antibody (Genetex) diluted 1:500.

Techniques: Expressing

Journal: Carcinogenesis

Article Title: Granulin-epithelin precursor interacts with heparan sulfate on liver cancer cells

doi: 10.1093/carcin/bgu164

Figure Lengend Snippet: Purity and signaling transduction of rGEP. ( A ) Purified rGEP was either untreated or deglycosylated by PNGase F and was analyzed in SDS–PAGE. Left panel shows the visualization of Coomassie blue staining (CB), where PNGase F was found at ~30kDa. The middle and right panels show the western blot (WB) analysis after detection of anti-His antibody and GEP mAb, respectively. One microgram of rGEP was used for each lane in Coomassie blue staining, whereas 10ng was used for western blot. ( B ) HepG2 and the GEP-suppressed Hep3B-sh1 were FBS-starved for 48h and were incubated with rGEP for 5min, followed by trypsin-EDTA detachment, formaldehyde fixation and 70% methanol permeabilization. Specific antibodies against pAKT and pERK1/2 were used for detection. If inhibitors were involved, 100nM wortmannin (W) or 10 µM U0126 (U) was added to the cells 1h in prior to the assay and was withdrawn before rGEP incubation. Representative histograms with isotypic controls (filled area), untreated samples (black line) and cells treated with 0.4 µg/ml rGEP (red line) are shown. The geometric mean fluorescent intensity (MFI) of each sample is shown in the graph. In parallel with rGEP, EGF was used as a positive control for the activation of signaling pathways (data not shown). Asterisks represent significant differences from the control without adding rGEP at 95% level according to Student’s t -test.

Article Snippet: Anti-GEP mAb generated against the GEP carboxyl-terminus ( ) (Versitech, Hong Kong) was used in rGEP binding neutralization.

Techniques: Transduction, Purification, SDS Page, Staining, Western Blot, Incubation, Positive Control, Activation Assay, Protein-Protein interactions, Control

Journal: Carcinogenesis

Article Title: Granulin-epithelin precursor interacts with heparan sulfate on liver cancer cells

doi: 10.1093/carcin/bgu164

Figure Lengend Snippet: Binding properties of rGEP on liver cancer cell lines. ( A ) Confocal images of rGEP binding on HCC cell surface. Hep3B and HepG2 were incubated with rGEP, specific GEP antibody, anti-rabbit second antibody and Texas Red-conjugated WGA. The cells were then fixed, permeabilized and stained with 4′,6-diamidino-2-phenylindole. ( B ) Cell surface-binding capacity of Hep3B (left panel) and HepG2 (right panel) for rGEP. Detached cells were incubated with different amounts (0.4–3.2 µg) of rGEP at 4°C and the cell surface binding of rGEP was detected by FITC-anti-His antibody. The histograms show fluorescent intensity of cells and the shaded peaks are isotypic controls. The bar charts show the geometric mean fluorescent intensity (MFI) of different samples. ( C ) Neutralization of rGEP binding by GEP mAb (×GEP). GEP mAb or mouse IgG were premixed with 0.8 µg rGEP. This rGEP-antibody mixture was added to EDTA-detached Hep3B for binding. The histogram shows the fluorescent intensity of cells bound with pretreated rGEP. The bar chart shows the percentage of cells in the positive region in different samples. Untreated cells detected by isotypic control antibody (IC) and anti-His antibody (basal) are also shown. ( D ) Competitive binding assay of rGEP. Different growth factors were preincubated with detached Hep3B at 4°C, followed by the binding of 0.8 µg rGEP, detection of anti-His antibody and quantification in flow cytometry. The bar chart shows the percentage of cells in the positive region where the IC and the anti-His basal levels are also shown. The percentages shown are the means and standard deviations obtained from three independent assays. Asterisks represent significant differences from the ‘rGEP only’ at 95% level according to Student’s t -test.

Article Snippet: Anti-GEP mAb generated against the GEP carboxyl-terminus ( ) (Versitech, Hong Kong) was used in rGEP binding neutralization.

Techniques: Binding Assay, Incubation, Staining, Neutralization, Control, Competitive Binding Assay, Flow Cytometry

Journal: Carcinogenesis

Article Title: Granulin-epithelin precursor interacts with heparan sulfate on liver cancer cells

doi: 10.1093/carcin/bgu164

Figure Lengend Snippet: GEP interacts with HS on cell surface of liver cancer cell line. ( A – C ) Hep3B cells were incubated with the indicated enzymes in lyase buffer at 37°C for an hour. Cells were detached by EDTA and collected for antibody detection and rGEP binding. The bar charts show the geometric mean fluorescent intensity (MFI) of each sample, whereas ‘basal’ represents untreated cells detected by anti-His without the addition of rGEP. Because the basal MFI between treated cells and untreated cells are similar, only that of the untreated cells is shown. The histograms show the peaks of untreated cells and cells treated with 0.8 mIU/ml HepIII, whereas shaded peak represents isotypic control (IC). (A) Cell surface HS of the detached cells was detected by HS mAb. (B) The treated cells were detected by FITC-conjugated GEP mAb to assess the change of cell surface expression of GEP. (C) The detached cells were incubated with 0.8 µg rGEP at 4°C to determine their rGEP binding capacity. The bound rGEP was then detected by anti-His antibody. ( D ) Hep3B cells were detached and incubated with 0.8 µg rGEP at 4°C. The cells were then incubated with the indicated amounts of heparin on ice for 15min and then washed with blocking buffer for twice while the residual bound rGEP was detected by anti-His antibody. In the histogram, only the IC, untreated cells and the sample washed by 10 µg/ml heparin are shown. Chond: chondroitinase ABC; HepIII: heparinase III; IU: international unit.

Article Snippet: Anti-GEP mAb generated against the GEP carboxyl-terminus ( ) (Versitech, Hong Kong) was used in rGEP binding neutralization.

Techniques: Incubation, Binding Assay, Control, Expressing, Blocking Assay

Journal: Carcinogenesis

Article Title: Granulin-epithelin precursor interacts with heparan sulfate on liver cancer cells

doi: 10.1093/carcin/bgu164

Figure Lengend Snippet: Mapping of heparin-binding domain in GEP and the contribution of the heparin-binding domain in HCC cell surface binding. ( A ) Schematic diagram of GEP protein, rGEP and derivatives and three granulin subunits. The seven and half granulins are shown as grey boxes in the GEP protein. Open arrows represent the N-glycosylation sites identified previously. The locations of rGEP, the deletion mutants (N492, C101, C77 and C51), the derivatives (rGEP-2A, rGEP-3A and rGEP-5A) and three granulin proteins corresponding to the GEP protein are shown. ( B and C ) Immunoblotting after heparin sepharose chromatography. The rGEP, deletion mutants, derivatives and granulin proteins were collected from media of the transfected COS-1. These media (P) were applied to heparin sepharose for incubation. The flow through (FT) was collected after 30min incubation. The sepharose was washed by Tris buffer for twice (W 1 and W 2 ). Elution buffer containing 0.125–2M NaCl was applied sequentially to the sepharose. The recombinant proteins were then detected by anti-His antibody in the immunoblotting. ( D ) Purified rGEP-3A and rGEP derivatives N492 and C101 were analyzed in SDS–PAGE. Protein was stained by Coomassie blue (CB) or detected by anti-His antibody in western blot (WB). ( E ) Polymerization of rGEP is independent of heparin and heparin-binding domain. Different amounts of heparin (0–100 µg/ml) were incubated with the recombinant protein at room temperature with or without DSS cross-linking. The polymerization status of rGEP (lanes A–F) and rGEP-3A (lanes G-L) was assessed by SDS–PAGE analysis and immunoblotting detected by anti-His antibody. ( F ) Indicated amounts of purified rGEP, rGEP-3A, N492 and C101 were incubated with detached Hep3B to compare their binding ability. The binding was then detected by FITC-anti-His antibody. In the case of HS depletion, cells were incubated with heparinase III in lyase buffer at 37°C for an hour before cell detachment. The histogram shows the fluorescent signal of the cells when incubate with 0.8 µg rGEP, N492 and C101. The shaded area represents isotypic control. The bar chart shows the geometric mean fluorescent intensity (MFI) of the cells when incubated with different amount of proteins.

Article Snippet: Anti-GEP mAb generated against the GEP carboxyl-terminus ( ) (Versitech, Hong Kong) was used in rGEP binding neutralization.

Techniques: Binding Assay, Glycoproteomics, Western Blot, Chromatography, Transfection, Incubation, Recombinant, Purification, SDS Page, Staining, Control

Journal: Carcinogenesis

Article Title: Granulin-epithelin precursor interacts with heparan sulfate on liver cancer cells

doi: 10.1093/carcin/bgu164

Figure Lengend Snippet: rGEP binding and rGEP-mediated signaling transduction in HCC cells after suppression of GPC3 and EXT1. Cells were transfected with NC siRNA and siRNAs targeting GPC3 and EXT1 (HSS104149 and HSS103435, respectively, from Life Technologies). ( A ) The cells were detached at 48h posttransfection for rGEP binding assay. The detached cells were incubated with 3.2 µg rGEP, followed by detection of anti-His antibody and analysis in flow cytometry. The chart shows the relative percentage of binding by comparing the MFI of each transfectant to the untreated cells (P). The means and the standard deviations were obtained from three independent assays. Asterisks represent significant differences from the ‘NC’ at 95% level according to Student’s t -test. The binding properties of Hep3B and Hep3B-sh1 are the same. ( B ) Suppression of GPC3 and EXT1 in HCC cell reduced the rGEP-mediated signaling transduction. Two cell lines (HepG2 and GEP-suppressed Hep3B) were transfected with the siRNAs and were treated with or without 0.2 µg/ml rGEP for 5min. Cells were detached, fixed by formaldehyde, permeabilized by 70% methanol and stained with fluorescein-conjugated specific antibody against pAKT and pERK1/2. The graphs show the difference of MFI with and without rGEP induction, reflecting the increased amount of pAKT and pERK1/2 upon rGEP induction in different siRNA treated cells. Asterisks represent significant differences from the ‘NC’ at 95% level according to Student’s t -test.

Article Snippet: Anti-GEP mAb generated against the GEP carboxyl-terminus ( ) (Versitech, Hong Kong) was used in rGEP binding neutralization.

Techniques: Binding Assay, Transduction, Transfection, Incubation, Flow Cytometry, Staining