FIGURE 2.

LBR specifically bind to H4K20me2. A, histone peptide array. Celluspot was incubated with 100 nm of GST-NP^WT and probed with anti-GST antibody. The spots were detected by chemiluminescence. Detected spots related to H4K20 modifications are shown (see supplemental Fig. S1 and Table S1 for complete data). B, GST-NP^WT and -NP^W16A beads incubated with recombinant histone H4, either unmodified (Un), monomethylated at Lys²⁰ (me1) or dimethylated at Lys²⁰ (me2), washed, and then subjected to SDS-PAGE. Bound H4 was detected by modification-independent anti-H4 antibody. GST and GST-NP fragments on beads are shown in the lower panel. C, ChIP. PANC1 cells (5.0 × 10⁶) were cross-linked with paraformaldehyde, and the chromatin was digested by sonication and micrococcal nuclease until most of the chromatin became a mononucleosome. After immunoprecipitation by the indicated antibodies (normal IgG and LBR), the beads were washed and reverse cross-linked by heating, and then the histone modification profile was analyzed by Western blotting (WB), indicated on the right. Inputs were 0.5, 0.25, and 0.05%, respectively. D, H4K20me2 modification enriched beneath the NE. HeLa cells were fixed and immunostained with anti-H4K20me2 and -LBR antibodies. The boxed area is enlarged at the bottom right. Arrows indicate the NE. Scale bar, 20 μm.