rabbit polyclonal anti-hbc antibody  (Austral Biologicals)

Journal: mSphere

Article Title: Exploring the tolerable region for HiBiT tag insertion in the hepatitis B virus genome

doi: 10.1128/msphere.00518-24

Figure Lengend Snippet: Detection of HBcAg and HiBiT-tagged viral proteins by transfection of HiBiT-tagged HBVcc plasmids. ( A ) The production of HBcAg and HiBiT-tagged viral proteins was detected by immunostaining with anti-HBc and anti-HiBiT antibodies 3 days after transfection. Nuclei were visualized by staining with DAPI. The white bar indicates 100 µM. ( B ) The HiBiT signals in cells and culture medium were measured after transfection of plasmids for HiBiT-tagged HBVcc. HBVcc-WT was used as a negative control. ( C ) HBsAg production in the culture medium was measured after transfection of plasmids for HiBiT-tagged HBVcc.

Article Snippet: The expression of HBcAg in transfected cells was detected by staining with a rabbit polyclonal anti-HBc antibody (Beacle Inc., Kyoto, Japan) and Alexa Fluor 555-conjugated anti-rabbit IgG (Thermo Fisher Scientific).

Techniques: Transfection, Immunostaining, Staining, Negative Control

Journal: mSphere

Article Title: Exploring the tolerable region for HiBiT tag insertion in the hepatitis B virus genome

doi: 10.1128/msphere.00518-24

Figure Lengend Snippet: Infection of HiBiT-tagged HBVcc in human primary hepatocytes. ( A ) Human primary hepatocytes were infected with HiBiT-tagged HBVcc at 200 GEq/cell, and the infection efficiencies were compared with those of HBVcc-WT by monitoring HBsAg and HiBiT signals in culture medium on the indicated days after infection. ( B ) The HBV DNA levels in the culture media of HBVcc-WT- and HiBiT-tagged HBVcc-infected cells were assessed by real-time PCR with a primer and probe set designed to target the HBs region after treatment with DNase after 12 days of culture. ( C ) HBVcc-WT- and HiBiT-tagged HBVcc-infected cells were detected by staining with rabbit polyclonal anti-HBc antibody and Alexa Fluor 555-conjugated anti-rabbit IgG. Nuclei were visualized by staining with DAPI.

Article Snippet: The expression of HBcAg in transfected cells was detected by staining with a rabbit polyclonal anti-HBc antibody (Beacle Inc., Kyoto, Japan) and Alexa Fluor 555-conjugated anti-rabbit IgG (Thermo Fisher Scientific).

Techniques: Infection, Real-time Polymerase Chain Reaction, Staining

Journal: mSphere

Article Title: Exploring the tolerable region for HiBiT tag insertion in the hepatitis B virus genome

doi: 10.1128/msphere.00518-24

Figure Lengend Snippet: Infection of HepG2/NTCP cells with HiBiT-tagged HBVcc.( A ) HepG2/NTCP cells were infected with HiBiT-tagged HBVcc at 200 GEq/cell, and the infection efficiencies were compared with those of HBVcc-WT by monitoring HBsAg and HiBiT signals in the culture media on the indicated days after infection. ( B ) The HBV DNA levels in the culture media of HBVcc-WT- and HiBiT-tagged HBVcc-infected cells were assessed by real-time PCR with a primer and probe set designed to target the HBs region after treatment with DNase after 12 days of culture. ( C ) HBVcc-WT- and HiBiT-tagged HBVcc-infected cells were detected by staining with rabbit polyclonal anti-HBc antibody and Alexa Fluor 555-conjugated anti-rabbit IgG. Nuclei were visualized by staining with DAPI.

Article Snippet: The expression of HBcAg in transfected cells was detected by staining with a rabbit polyclonal anti-HBc antibody (Beacle Inc., Kyoto, Japan) and Alexa Fluor 555-conjugated anti-rabbit IgG (Thermo Fisher Scientific).

Techniques: Infection, Real-time Polymerase Chain Reaction, Staining

Journal: PLoS ONE

Article Title: C-Terminal Substitution of HBV Core Proteins with Those from DHBV Reveals That Arginine-Rich 167 RRRSQSPRR 175 Domain Is Critical for HBV Replication

doi: 10.1371/journal.pone.0041087

Figure Lengend Snippet: (A) Schematic diagrams of HBV, DHBV, and chimeric C protein variant constructs aligned with amino acid sequences of HBV and DHBV C protein carboxyl-terminal domains. Amino acids in bold are identical or homologous. SRPK and PKA phosphorylation sites of HBV are marked with asterisks and arrowheads, respectively. Phosphorylation sites of DHBV , are marked with open arrowheads. Amino acid sequences of the HBV and DHBV C proteins are presented as open and closed boxes, respectively. The cytomegalovirus immediate early (CMV IE) promoter is represented as an open arrow. PRE, post-transcriptional regulatory element. (B) Identification of C protein and core particles by chimeric C protein variants. To examine expression of C protein variants, lysates from HuH7 cells transfected with a pHCP, pDCP, pHD192–262, pHD192–220, pHD221–262, pHCP145, pHCP145–R127Q, or C-deficient mutant were electrophoresed on 12% SDS-PAGE gels and protein levels visualized by Western blotting using polyclonal rabbit anti-HBc antibody (top panel). C protein variants (arrowheads) with expected molecular weights are indicated. The C-deficient mutant lacks C protein due to the introduction of a stop codon at Glu 8 in the C ORF. The pHCP and the C-deficient mutant constituted positive and negative controls, respectively. Transfection experiments were repeated four times. To detect core particles formed by C protein variants from native agarose gels, isolated core particles were transferred to PVDF membranes and incubated with polyclonal rabbit anti-HBc antibody (second panel). The Renilla luciferase expression plasmid phRL-CMV was co-transfected into HuH7 cells as a transfection control (third panel). Luciferase and α-tubulin (bottom panel) levels were determined by Western blotting using polyclonal rabbit anti-luciferase and monoclonal mouse anti-tubulin antibodies as transfection and loading controls, respectively. HRP-conjugated secondary antibody and enhanced chemiluminescence were used to visualize C, α-tubulin, and luciferase proteins and core particles. (C) Relative levels of C protein expression and core particle assembly by chimeric C protein variants. Relative levels of C proteins, core particles, and luciferase were measured with the Fujifilm Image Gauge V4.0 program. Relative levels of C protein variant expression and core particle assembly were compared to normalized transfection efficiencies. The data represent the mean ± standard deviation (SD) from four independent experiments.

Article Snippet: For detection of core particles, both polyclonal rabbit anti-HBc antibody (diluted 1∶1000; DAKO, Carpinteria, CA, USA) and our antibody (diluted 1∶1000) were used interchangeably ( ).

Techniques: Variant Assay, Construct, Expressing, Transfection, Mutagenesis, SDS Page, Western Blot, Isolation, Incubation, Luciferase, Plasmid Preparation, Standard Deviation

Journal: Virus Research

Article Title: Identification of neutralizing epitopes in the preS2 domain of the hepatitis B virus

doi: 10.1016/j.virusres.2022.199014

Figure Lengend Snippet: Immunization of mice with plasmids encoding HBV preS1 and preS2. (A) Schematic diagram of the structure of HBsAg and of plasmids encoding N-terminal preS1 and preS2, showing the position of the CAG promoter (CAG) and of sequences encoding the murine Ig κ-chain leader sequence (S), Myc tag (myc), spacer, and GroEL. The amino acid sequences of preS1/2–47 and preS2 are derived from HBV genotype-C. (B) Expression of preS1/2–47 and preS2. Lysates and supernatants of 293T cells transfected with either plasmid were harvested at 3 days post-transfection and subjected to immunoblotting using anti-preS1 and anti-preS2; anti-actin antibodies were used as a loading control. (C) Detection of serum antibodies binding to the preS1/2–47 peptide, preS2 peptide, HBs-L. HBs-M, and HBs-S proteins. Individual sera from mice (n=4) immunized with the indicated plasmids were diluted 100-fold for peptides and 250-fold for HBs proteins; the diluted sera then were added to ELISA microtiter wells containing each antigen. Bound antibody was detected using HRP-conjugated anti-mouse secondary antibody. The vertical axis for each serum and antigen shows absorbance in individual wells. (D and E) Neutralizing activities of sera from mice immunized with preS1 (D) or preS2 (E) expression plasmids. HBV/NL derived from genotype-C was preincubated with pooled sera (at 100-, 200-, 400-, 800-, and 1600-fold dilutions) derived from mice immunized with the indicated plasmids; the mixtures then were then used to infect G2/NT-18C cells for 16 h. Luciferase activity was determined at 7 days post-infection and is expressed relative to activity in reactions without serum. The statistical significance of differences between groups was evaluated using a Student's t-test (* P < 0.05, ** P < 0.01 vs. serum from empty vector-immunized control group). (F) HBVcc was mixed with indicated serum (100-fold dilutions) and used to inoculate HepG2-NTCPsec+ cells. HBsAg titers in the culture medium were measured. Statistical significance was evaluated using the Student's t-test (** P < 0.01 vs. no serum control). (G) HBV-positive cells were visualized by staining with an anti-HBc antibody, and nuclei were visualizedby staining with DAPI.

Article Snippet: The HBVcc-infected cells were fixed, permeabilized, and treated with rabbit polyclonal anti-HBc IgG (Austral Biologicals) followed by staining with Alexa Fluor 555-conjugated anti-rabbit IgG (Thermo Fisher Scientific).

Techniques: Sequencing, Derivative Assay, Expressing, Transfection, Plasmid Preparation, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Infection, Staining

Journal: Virus Research

Article Title: Identification of neutralizing epitopes in the preS2 domain of the hepatitis B virus

doi: 10.1016/j.virusres.2022.199014

Figure Lengend Snippet: Neutralization assay of anti-preS2 mAbs using HBV/NL derived from HBV genotype-C. (A) mAbs (0.5, 1, and 5 μg/mL) were incubated with HBV/NL (20 GEq/cell) for 1 h; the mixtures then were used to infect G2/NT-18C cells for 16 h. Luciferase activity of cells was determined at 7 days post-infection and is expressed relative to activity in cells infected with reactions performed without antibodies. Values represent the means of quadruplicate reactions; error bars indicate standard deviations. Statistical significance was evaluated using the Student's t-test (** P < 0.01 vs. no antibody control). (B) HBVcc was mixed with the indicated mAbs (1 μg/mL) and used to inoculate HepG2-NTCPsec+ cells. HBsAg titers in the culture medium were measured. Statistical significance was evaluated using the Student's t-test (** P < 0.01 vs. no antibody control). (C) HBV-positive cells were visualized by staining with an anti-HBc antibody; nuclei were visualized by staining with DAPI. (D) Epitope mapping of mAbs against preS2. Minimum epitopes recognized by each mAb are shown. Thirty-six synthetic overlapping 20-aa peptides corresponding to preS2 were used. The horizontal axis in each of the mAb panels shows absorbance units. The peptides showing antibody binding are indicated by an asterisk.

Article Snippet: The HBVcc-infected cells were fixed, permeabilized, and treated with rabbit polyclonal anti-HBc IgG (Austral Biologicals) followed by staining with Alexa Fluor 555-conjugated anti-rabbit IgG (Thermo Fisher Scientific).

Techniques: Neutralization, Derivative Assay, Incubation, Luciferase, Activity Assay, Infection, Staining, Binding Assay

Journal: Pharmaceuticals

Article Title: Design and Synthesis of Hepatitis B Virus (HBV) Capsid Assembly Modulators and Evaluation of Their Activity in Mammalian Cell Model

doi: 10.3390/ph15070773

Figure Lengend Snippet: Analysis of the effects of HAP compounds on assembly of HBV capsids in mammalian cells. BHK-21 cells were infected with SFV1/HBc (D1 genotype) to express full-length HBc gene. After 4 h, the cell culture medium was replaced with compound-containing medium, and after 24 h of cell incubation with respective compound the medium was removed and the cell lysates were analyzed by native agarose gel electrophoresis and subsequent anti-HBc immunoblot. ( A ) Cells treated with compounds: 1a (Bay 41-4109), 10 µM; 1b , 10 µM; 1g , 10 µM; 1f , 10 µM; 1h , 10 µM; 1i , 10 µM; 1e , 10 µM; 1d , 10 µM; HBc , non-treated HBc producing BHK-21 control cells; Lam (lamivudine), 10 µM; 3a , 20 µM; 3b , 20 µM; 6 , 10 µM. ( B ) Cells treated with compounds: HBc , non-treated HBc producing BHK-21 control cells; 3d , 50 µM; 3c , 50 µM; 1k , 10 µM; 4b , 50 µM; 7a , 25 µM; 7b , 1 µM; 1c , 5 µM; 1j , 5 µM; 4a , 5 µM; 5 , 50 µM. Capsid bands are indicated with arrows; capsid-like structures are indicated with brace. Equal protein amount of the total cell lysates was loaded in each well of the agarose gel. Compounds selected for further dose-dependent study are indicated in red circles.

Article Snippet: This specific pattern was also observed by performing staining with rabbit polyclonal anti-HBc antibodies (Dako, cat. No. B058601), data not shown.

Techniques: Infection, Cell Culture, Incubation, Agarose Gel Electrophoresis, Western Blot

Journal: Pharmaceuticals

Article Title: Design and Synthesis of Hepatitis B Virus (HBV) Capsid Assembly Modulators and Evaluation of Their Activity in Mammalian Cell Model

doi: 10.3390/ph15070773

Figure Lengend Snippet: Dose-dependent effects of selected compounds on the assembly of HBV capsids in mammalian cells. Native agarose gel electrophoresis and subsequent anti-HBc immunoblotting of the cell lysates is shown. BHK-21 cells were infected with SFV1/HBc (D1 genotype) to express full length HBc gene. After 4 h, the cell culture medium was replaced with compound-containing medium, and after 24 h of incubation the cell medium was removed and the lysates were loaded on agarose gel. Equal total protein amount was tested. The concentrations (in a range 0.1–50 µM) are indicated on the top of each immunoblot. The concentration “0” (without compound) for comp. 1b test corresponds to the line “0” in comp. 1h immunoblot; the concentration “0” for comp. 1f test corresponds to the line “0” in comp. 1d immunoblot, because it was the same run for both compounds with shared untreated HBc control.

Article Snippet: This specific pattern was also observed by performing staining with rabbit polyclonal anti-HBc antibodies (Dako, cat. No. B058601), data not shown.

Techniques: Agarose Gel Electrophoresis, Western Blot, Infection, Cell Culture, Incubation, Concentration Assay

Journal: Pharmaceuticals

Article Title: Design and Synthesis of Hepatitis B Virus (HBV) Capsid Assembly Modulators and Evaluation of Their Activity in Mammalian Cell Model

doi: 10.3390/ph15070773

Figure Lengend Snippet: Immunostaining of HBc protein in BHK-21 cells after treatment with HAP compound 1d . BHK cells were infected with recombinant SFV1/HBc virus, next day the cell culture medium was replaced with comp. 1d containing medium and incubated for 24h. After incubation the anti-HBc immunostaining analysis was performed using monoclonal C1-5 antibodies (green fluorescence). The cells were analyzed by confocal microscopy and compared with untreated cells. The nuclei were stained with DAPI (blue). ( A ) cells treated with 20 µM comp. 1d . ( B ) cells treated with 50 µM comp. 1d . ( C ) Untreated cells; the orange arrows indicate the nuclear localization of HBc in untreated cells. ( D ) Negative control represents BHK-21 cells uninfected with SFV1/HBc virus and untreated with the compound.

Article Snippet: This specific pattern was also observed by performing staining with rabbit polyclonal anti-HBc antibodies (Dako, cat. No. B058601), data not shown.

Techniques: Immunostaining, Infection, Recombinant, Cell Culture, Incubation, Fluorescence, Confocal Microscopy, Staining, Negative Control

Journal: Pharmaceuticals

Article Title: Design and Synthesis of Hepatitis B Virus (HBV) Capsid Assembly Modulators and Evaluation of Their Activity in Mammalian Cell Model

doi: 10.3390/ph15070773

Figure Lengend Snippet: Electron microscopy analysis of particles concentrated from the lysates of BHK-21 cells after treatment with comp. 1d . BHK-21 cells were infected with pSFV1/HBc to allow HBc protein expression. Three hours post-infection, 50 μM comp. 1d was added to cell medium, and the cells were further incubated for 24 h. After incubation the cell lysates were performed and used for ultracentrifugation through 20% sucrose cushion. The pellets containing capsids and high molecular capsid-like structures were resuspended and used for electron microscopy analysis (TEM). ( A ) Electron microscopy pictures of regular HBV core particles of standard T3 and T4 symmetry isolated from untreated cells (on the left) and irregular misassembled core particles isolated from cells treated with comp. 1d (on the right). ( B ) Selected HBc protein assembly products visible by electron microscopy from cells treated with HAP comp. 1d . The approximate particle size was measured as the longer and shorter diameter of the irregular form of the particle. ( C ) Native agarose anti-HBc immunoblot of samples prepared for electron microscopy: line 1—untreated HBc; line 2—HBc treated with comp. 1d .

Article Snippet: This specific pattern was also observed by performing staining with rabbit polyclonal anti-HBc antibodies (Dako, cat. No. B058601), data not shown.

Techniques: Electron Microscopy, Infection, Expressing, Incubation, Isolation, Western Blot

Journal: Frontiers in Microbiology

Article Title: The HBV Core Protein and Core Particle Both Bind to the PPiase Par14 and Par17 to Enhance Their Stabilities and HBV Replication

doi: 10.3389/fmicb.2021.795047

Figure Lengend Snippet: Par14 and Par17 are novel binding partners of HBc and the core particle. (A) Co-immunoprecipitation reveals that Par14 and Par17 directly interact with the core particle and/or HBc of HBV. Huh7 cells in 6 cm plates were mock-transfected (lane 1) or co-transfected with 4 μg of Myc-HBc WT plus 3 × FLAG (lanes 2, 5, and 8), 3 × FLAG-Par14 WT (lanes 3, 6, and 9), or 3 × FLAG-Par17 WT (lanes 4, 7, and 10). At 72 h post-transfection, whole-cell lysates were prepared (lanes 1–4) and immunoprecipitated with an anti-Myc or anti-FLAG antibody (lanes 5–7). As a negative control, lysates were immunoprecipitated with normal rabbit IgG (Merck Millipore #12–370) or normal mouse IgG (Merck Millipore #12–371; lanes 8–10). SDS-PAGE and immunoblotting were performed. Resolved proteins were transferred to PVDF membranes and incubated overnight with mouse monoclonal anti-FLAG M2 (Sigma #F1804), rabbit monoclonal anti-PIN4 (1:1,000, Abcam #ab155283), rabbit polyclonal anti-Myc (Santa Cruz Biotech #sc-789), rabbit polyclonal anti-HBc (1:1,000, 17), and mouse monoclonal anti-GAPDH (1:5,000, Santa Cruz #sc-32,233) primary antibodies. GAPDH was used as a loading control. The blots were incubated with secondary antibodies (anti-mouse or anti-rabbit) coupled to horseradish peroxidase (1,5,000 dilution, Thermo Fisher Scientific). (B) NAGE and core particle immunoblotting reveal that the core particle of HBV interacts with Par14/Par17. Huh7 cells in 6 cm plates were mock-transfected (lane 1) or transfected with 3 × FLAG-Par14 WT (lane 3) or 3 × FLAG-Par17 WT (lane 4) or co-transfected with Myc-HBc WT plus 3 × FLAG (lane 2), 3 × FLAG-Par14 WT (lane 5), or 3 × FLAG-Par17 WT (lane 6). At 72 h post-transfection, cell lysates were prepared and subjected to 1% NAGE, transferred to a PVDF membrane, and immunoblotted with anti-FLAG, anti-PIN4, anti-Myc, and anti-HBc antibodies. (C,D) Both the HBV core particle and HBc interact with Par14/Par17. (C) NAGE and core particle immunoblotting demonstrate that HBc-Y132A is core particle assembly-defective, unlike HBc WT. Huh7 cells were mock-transfected (lane 1) or co-transfected with Myc-HBc WT or core particle assembly-defective Myc-HBc-Y132A plus 3 × FLAG (lanes 2 and 5), 3 × FLAG-Par14 WT (lanes 3 and 6), or 3 × FLAG-Par17 WT (lanes 4 and 7). (D) Co-immunoprecipitation reveals that Par14/Par17 directly interact with core particle assembly-defective HBc-Y132A. Lysates of transfected Huh7 cells were immunoprecipitated and subjected to SDS-PAGE as described above. The immunoblots were visualized by enhanced chemiluminescence (ECL Western blotting detection reagent, Amersham). Endogenous Par14 is marked with an arrow. Overexpressed Par14 and Par17 are marked with a double arrowhead and open arrowhead, respectively. A representative result from three independent experiments is shown.

Article Snippet: Crosslinked sonicated chromatin was subjected to immunoprecipitation with 3 μg of rabbit monoclonal anti-PIN4 (Abcam #ab155283), mouse monoclonal anti-FLAG M2 (Sigma #F1804), rabbit polyclonal anti-HBc , mouse monoclonal anti-RNA polymerase II (Abcam #ab817), rabbit polyclonal anti-AcH3 (Merck Millipore #06–599), and rabbit polyclonal anti-H3 (Abcam #ab1791) antibodies or normal mouse or rabbit IgG (negative controls) for 16 h at 4°C, incubated with protein A/G-plus agarose beads (Santa Cruz Biotechnology #sc-2003) overnight at 4°C, and centrifuged at 1,000 g for 5 min at 4°C to recover immunoprecipitated protein–DNA complexes.

Techniques: Binding Assay, Immunoprecipitation, Transfection, Negative Control, SDS Page, Western Blot, Incubation, Control, Membrane

Journal: Frontiers in Microbiology

Article Title: The HBV Core Protein and Core Particle Both Bind to the PPiase Par14 and Par17 to Enhance Their Stabilities and HBV Replication

doi: 10.3389/fmicb.2021.795047

Figure Lengend Snippet: The S19 and E46/D74 residues of Par14 and S44 and E71/D99 residues of Par17 promote recruitment of HBc into cccDNA via the HBc RP motif. (A) Overexpressed Par14 and Par17 increase recruitment of HBc into cccDNA. Vector- (lane 2), Par14- (lane 3), and Par17-transduced (lane 4) HepG2-hNTCP-C9 cells were infected with 1.7 × 10 3 GEq of HBV WT. Mock-infected HepG2-hNTCP-C9 cells were used a negative control (lane 1). (B) PIN4 KD reduces binding of HBc to cccDNA. HepG2-hNTCP-C9-shControl (lane 3), HepG2-hNTCP-C9-shPIN4-#1 (lane 4), and HepG2-hNTCP-C9-shPIN4-#5 (lane 5) cells were infected with HBV as described above. HepG2 cells were infected (lane 1) and HepG2-hNTCP cells were mock-infected (lane 2) as negative controls. (C,D) Substrate- and DNA-binding residues of Par14 (E46/D74 and S19, respectively) and Par17 (E71/D99 and S44, respectively) are important for enhanced recruitment of HBc into cccDNA. HepG2-hNTCP-C9 cells were plated as described above and mock-transfected (lane 1) or transfected with 3 × FLAG (lane 2), 3 × FLAG-Par14 WT (lane 3), 3 × FLAG-Par14-E46A/D74A (lane 4), 3 × FLAG-Par14-S19E (lane 5), 3 × FLAG-Par17 WT (lane 6), 3 × FLAG-Par17-E71A/D99A (lane 7), or 3 × FLAG-Par17-S44E (lane 8) (C) . HepG2-hNTCP-C9 (lane 1), HepG2-hNTCP-C9-shControl (lane 2), and HepG2-hNTCP-C9-shPIN4-#1 (lanes 3–10) cells were seeded as described above. As additional controls, HepG2-hNTCP-C9-shPIN4-#1 cells were mock-transfected (lane 3) or transfected with 3 × FLAG (lane 4). HepG2-hNTCP-C9-shPIN4-#1 cells were transfected with 3 × FLAG-Par14 WT (lane 5), 3 × FLAG-Par14-E46A/D74A (lane 6), 3 × FLAG-Par14-S19E (lane 7), 3 × FLAG-Par17 WT (lane 8), 3 × FLAG-Par17-E71A/D99A (lane 9), or 3 × FLAG-Par17-S44E (lane 10) (D) . (E,F) The HBc RP motif is critical for recruitment of HBc into cccDNA. HepG2-hNTCP-C9 (lane 1), HepG2-hNTCP-C9-shControl (lanes 2 and 3), and HepG2-hNTCP-C9-shPIN4-#1 (lanes 4 and 5) cells were infected with HBV WT (lanes 2 and 4) or HBV-HBc-AAP mutant (lanes 3 and 5) virions at 1.7 × 10 3 GEq per cell as detailed in the “Materials and Methods” section. (G) The HBc CTD is important for recruitment of HBc into cccDNA. HepG2-hNTCP-C9 (lane 1), HepG2-hNTCP-C9-shControl (lane 2), and HepG2-hNTCP-C9 PIN4 -KD (lane 3) cells were used as controls. HepG2-hNTCP-C9 PIN4 -KD cells were co-transfected with 3 × FLAG plus HBc WT (lane 4), the HBc CTD-deficient construct (lane 5), 3 × FLAG-Par14 WT plus HBc WT (lane 6) or the HBc CTD-deficient construct (lane 7), or 3 × FLAG-Par17 WT plus HBc WT (lane 8) or the HBc CTD-deficient construct (lane 9). HepG2-hNTCP-C9-shControl (lane 2) and HepG2-hNTCP-C9 PIN4 -KD (lanes 3–9) cells were infected with HBV as described above. At 9 days p.i., chromatin solutions were prepared as described previously [35] and subjected to immunoprecipitation with an anti-PIN4, anti-FLAG, anti-HBc, anti-RNA polymerase II (Abcam #ab817), anti-acetyl H3 (Merck Millipore #06–599), or anti-H3 antibody (positive control) or normal rabbit polyclonal IgG (negative control). Immunoprecipitated chromatin was analyzed by semi-quantitative PCR (A–E,G) or quantitative real-time PCR (F) . Relative levels of HBc, RNA polymerase II, acetyl H3, and HBV RNA were measured using ImageJ 1.46r. Representative data from three independent experiments are shown. Statistical significance in E and F was evaluated using Student’s t -test. * p < 0.05 (E) and exact p -values (F) relative to the corresponding controls are shown.

Article Snippet: Crosslinked sonicated chromatin was subjected to immunoprecipitation with 3 μg of rabbit monoclonal anti-PIN4 (Abcam #ab155283), mouse monoclonal anti-FLAG M2 (Sigma #F1804), rabbit polyclonal anti-HBc , mouse monoclonal anti-RNA polymerase II (Abcam #ab817), rabbit polyclonal anti-AcH3 (Merck Millipore #06–599), and rabbit polyclonal anti-H3 (Abcam #ab1791) antibodies or normal mouse or rabbit IgG (negative controls) for 16 h at 4°C, incubated with protein A/G-plus agarose beads (Santa Cruz Biotechnology #sc-2003) overnight at 4°C, and centrifuged at 1,000 g for 5 min at 4°C to recover immunoprecipitated protein–DNA complexes.

Techniques: Plasmid Preparation, Infection, Negative Control, Binding Assay, Transfection, Mutagenesis, Construct, Immunoprecipitation, Positive Control, Real-time Polymerase Chain Reaction