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. 2020 Jan 21;11:419. doi: 10.1038/s41467-020-14299-9

Fig. 3. R75A S-HDAg mutation affects the binding to BAZ2B BRD without altering S-HDAg localization and acetylation.

Fig. 3

a Top panel: schematic representation of S-HDAg domains. HLH helix loop helix domain encompassing arginine-rich motifs (ARM), LZ leucine zipper-like polymerization domain, NLS nuclear localization signal, RBD RNA-binding domain. Middle panel: consensus alignment of 274 HDAg sequences displayed as a WebLogo® showing the K72ac-X-X-R75 motif (squared) with perfect conservation of the K72 and R75 residues. Bottom panel: alignment of H3 and H4 tails, hSNF2L and hSNF2H indicating the acetylated motif for each protein. b Wt S-HDAg and R75A S-HDAg proteins have a similar nuclear localization pattern. Huh7 cells, transfected with plasmids coding for either wt or R75A S-HDAg proteins, were subjected to indirect immunofluorescence at day 3, using an HDAg antibody (green), and nuclei were stained using DAPI (blue). Images were captured by confocal microscopy (objective ×63; digital zoom 0.8; bar = 10 μm). c Wt and R75A S-HDAg are expressed at the same level. Huh7 cells stably expressing wt or R75A S-HDAg proteins were subjected to subcellular fractionation. Wt and R75A S-HDAg levels in the nuclear (Nucl) and cytoplasmic (Cyto) fractions were determined by IB using the α-HDAg antibody. The α-Alpha Tubulin and α-Histone H3 antibodies verified fraction purity and loading amount. d Wt and R75A S-HDAg have similar acetylation levels in Huh7 cells stably expressing each protein. Equal quantities of nuclear protein extracts were immunoprecipitated with α-HDAg and immunoblotted with antibodies against acetyl-lysine. e Wt S-HDAg and R75A S-HDAg protein stability was compared in Huh7 cells treated with cycloheximide (CHX: 100 μg/ml). Left panel: Total cell extracts were analyzed by western blotting using the indicated antibodies. Right panel: Densitometric values expressed as ratio over wt S-HDAg or R75A S-HDAg controls (time 0). f Co-immunoprecipitation of S-HDAg and GFP-Tag-BAZ2B BRD. Parental Huh7 cells and Huh7 cells stably expressing wt or R75A S-HDAg were transfected with the pGFP-Tag-BAZ2B BRD plasmid coding for a GFP-BAZ2B BRD fusion protein targeted to the nucleus. Nuclear extracts were subjected to IP with GFP-Trap® beads. Input and elutions were analyzed by IB using the indicated antibodies. Source data are provided as a Source Data file.