FIGURE 7.
Identification of linker histone H1.5 isoform and HP1γ as NPM1-associated proteins. A, biochemical fractionation of proteins revealed that NPM1 is abundant in the chromatin fraction. Protein content in whole cell extracts (W) was compared with that obtained in a biochemical fractionation procedure using the same number of cells. Detergent-soluble proteins (Np), low-salt extracted nucleoplasmic proteins (Nu), and remaining chromatin (c)-bound proteins were separated. Histone H3 served as a marker for the chromatin fraction, and α-tubulin was used for the detergent-soluble protein fraction. B, U1242MG cells corresponding to 10 subconfluent p100 plates were subjected to nuclear complex co-IP. The immunoprecipitates were separated by SDS-PAGE, and the gels were subsequently stained with silver or CBB. IgH, immunoglobulin heavy chain; IgL, immunoglobulin light chain; Mw, molecular weight (kDa). Positions of IgH, IgL, and NPM1 are indicated. C, proteins extracted using the nuclear complex co-IP kit were immunoprecipitated with mouse monoclonal anti-NPM1 antibody overnight or an isotype IgG control antibody (IP Ctrl), and the material bound to beads was analyzed by SDS-PAGE followed by immunoblotting using anti-NPM1, anti-H3, anti-H3K9me3, anti-H3K9ac, anti-H1.5, and anti-HP1γ.