polyclonal rabbit anti-core antiserum  (Agilent technologies)

Journal: mBio

Article Title: Live Cell Imaging Reveals HBV Capsid Translocation from the Nucleus To the Cytoplasm Enabled by Cell Division

doi: 10.1128/mbio.03303-22

Figure Lengend Snippet: Changes to the subcellular distribution of HBV Cp over time. (A) Cartoon illustration of Cp divided into the N-terminal Assembly Domain and C-Terminal Domain (CTD). The CTD encodes the nuclear localization signal (NLS). The HBV Cp dimer crystal structure shown is based on PBD: 3J2V . The position of the Cp-Y132A mutation is highlighted in blue, shown on the red Cp subunit. (B) Outline of HBV expression and time course analysis workflow based on immunofluorescence (IF) detection of WT HBV Cp or Cp-Y132A. Images were developed using BioRender. (C) Representative images and quantification from IF time-course analysis of Huh7 cells expressing WT Cp with packageable pgRNAs, or the Cp-Y132 (no assembly) control. Cells were fixed at the indicated time points with WT Cp or Cp-Y132A detected using polyclonal anti-HBc antiserum. White dashed lines differentiate nuclei (N) from cytoplasm (C). Red dashed boxes highlight regions of interest, with red arrows indicating WT Cp puncta consistent with assembled capsids. Image scale bars represent 10 μm. Plots on the right present ratios of C/N mean fluorescence intensity (MFI) for 100 cells per condition per time point. The red dashed line at 1 indicates equivalent levels of nuclear and cytoplasmic fluorescence signals. Greater than 1 indicates more cytoplasmic MFI relative to the nucleus. Less than 1 indicates more nuclear MFI relative to the cytoplasm.

Article Snippet: For global Cp detection, a 1:1000 dilution of a polyclonal rabbit anti-core (anti-HBc) antiserum (Lot#:214-14, Austral Biologicals) was used prior to washing with PBS and staining with 1:1000 diluted secondary antibodies (goat anti-rabbit 488; Life Technologies).

Techniques: Mutagenesis, Expressing, Immunofluorescence, Fluorescence

Journal: mBio

Article Title: Live Cell Imaging Reveals HBV Capsid Translocation from the Nucleus To the Cytoplasm Enabled by Cell Division

doi: 10.1128/mbio.03303-22

Figure Lengend Snippet: Cp forms high-order assemblages in the nucleus. (A) Illustrations of Cp dimer and capsid structures highlighting the HBV Cp binding sites for the mAb3120 and polyclonal (anti-HBc) antibodies used for the dual labeling strategy. (B) Representative images from IF analysis of cells expressing WT Cp or Cp-Y132A, and incubated with the indicated antibodies, to confirm binding specificity. mCherry (inset, red) was co-transfected with the Cp variant to assist in identifying transfected cells prior to fixation, staining with DAPI (blue), and staining for IF (green). mAb3120 was confirmed as unable to detect Cp-Y132A (central panels). (C) Images and analysis of dually labeled Huh7 cells expressing WT Cp and detected using mAb3120 (cyan) and polyclonal anti-HBc (green). Transect analysis illustrates differential detection of Cp with polyclonal (predominantly nuclear) and mAb3120 (predominantly cytoplasmic) at 72h in a representative cell; consistent with unassembled Cp trafficking to the nucleus, even when the assembled capsid population is predominantly in the cytoplasm. Scale bars represent 10 μm. (D) Bar graphs quantifying the subcellular localization of WT Cp for 100 cells per condition, detected using the indicated antibody and corresponding to the experiment in (C). Error bars represent the standard deviation of the mean for 3 biological replicates. (E) Bar graphs as for (D) confirming a similar subcellular localization of WT Cp distribution over time when expressed from a plasmid encoding envelope glycoproteins (Env+).

Article Snippet: For global Cp detection, a 1:1000 dilution of a polyclonal rabbit anti-core (anti-HBc) antiserum (Lot#:214-14, Austral Biologicals) was used prior to washing with PBS and staining with 1:1000 diluted secondary antibodies (goat anti-rabbit 488; Life Technologies).

Techniques: Binding Assay, Labeling, IF-cells, Incubation, Transfection, Variant Assay, Staining, Expressing, Standard Deviation, Plasmid Preparation

Journal: mBio

Article Title: Live Cell Imaging Reveals HBV Capsid Translocation from the Nucleus To the Cytoplasm Enabled by Cell Division

doi: 10.1128/mbio.03303-22

Figure Lengend Snippet: Inducing cell cycle arrest using aphidicolin that entraps HBV Cp/capsids in the nucleus. Representative images showing Huh7 cells expressing WT HBV, treated with 10 μg/mL APC for the indicated time period (e.g., 24, 48, and 72 h post-gene expression). In the presence of APC, WT Cp remained predominantly nuclear at all time points, as detected by both capsid-specific mAb3120 and anti-HBc polyclonal antibody. Smaller images (top) bordered in red show WT HBV control WT Cp expression and relocalization from the nucleus to the cytoplasm in the absence of APC. Scale bars represent 10 μm. Bar graphs present data from 3 independent experiments, measuring 100 cells per condition and time point, with error bars representing the standard deviation of the mean.

Article Snippet: For global Cp detection, a 1:1000 dilution of a polyclonal rabbit anti-core (anti-HBc) antiserum (Lot#:214-14, Austral Biologicals) was used prior to washing with PBS and staining with 1:1000 diluted secondary antibodies (goat anti-rabbit 488; Life Technologies).

Techniques: Expressing, Standard Deviation

Journal: mBio

Article Title: Live Cell Imaging Reveals HBV Capsid Translocation from the Nucleus To the Cytoplasm Enabled by Cell Division

doi: 10.1128/mbio.03303-22

Figure Lengend Snippet: Increasing rates of capsid assembly does not affect Cp’s preferential accumulation in the nucleus at early time points. (A) Representative images of cells expressing rapid assembly mutant Cp-V124W tracked over a 72 h time course using dual label IF analysis. The red arrows indicate cytoplasmic co-localization of capsid/Cp detected by both mAb3120 and polyclonal anti-HBc antisera, respectively. Graphs present relative levels of nuclear versus cytoplasmic Cp-V124W distribution for 100 cells per time point. Error bars represent the standard deviation of the mean for 3 independent experiments. (B) Images from live single cell detection of fluorescent Cp-V124W-NG co-expressed with untagged Cp-V124W over an ~70 h time course. Time point labeled in red (28.5h) designates a cell division event. Please reference for the complete video file. Graph shows tracking of nuclear and cytoplasmic Cp-V124W-NG MFI for a representative cell, plotted over time. Gray box highlights an ~6 h time window encompassing a cell division event. (C) Images from IF detection of untagged WT Cp and Cp-V124W using polyclonal anti-HBc (green) with cellular nucleoli labeled using anti-Nucleophosmin (B23) (magenta) antibodies, indicating that both WT Cp and Cp-V124W localize to the nucleolus at early time points. Scale bars represent 10 μm. Red arrows highlight co-incident detection of Cp or Cp-V124W with Nucleophosmin (B23).

Article Snippet: For global Cp detection, a 1:1000 dilution of a polyclonal rabbit anti-core (anti-HBc) antiserum (Lot#:214-14, Austral Biologicals) was used prior to washing with PBS and staining with 1:1000 diluted secondary antibodies (goat anti-rabbit 488; Life Technologies).

Techniques: Expressing, Mutagenesis, Standard Deviation, Labeling