Prepatterning of differentiation-driven nuclear lamin A/C-associated chromatin domains by GlcNAcylated histone H2B

Assessment of specificity of the anti-H2BS112GlcNAc antibody. (A) Immunoreactivity of the anti-H2BS112GlcNAc antibody on H2BS112GlcNAc and corresponding unmodified H2B peptides (KHAVS¹¹²EGTK) immobilized on nitrocellulose. Peptides were preincubated without or with 2 ng/µL recombinant OGA prior to immunodetection. (B) Immunofluorescence peptide competition assay using H2BS112GlcNAc and unmodified H2B peptides; immunodetection using anti-H2BS112GlcNAc. Scale bar, 10 μm. (C) Immunoblot of ASC extracts preincubated with 0 or 2 ng/µL recombinant OGA for 30 min. (D) Expression of an EGFP-H2BS112A mutant, but not a control wild-type EGFP-H2B, in HeLa cells reduces H2BS112GlcNAc immunoreactivity (left) without affecting detection of total H2B (middle) or EGFP (right).