Fig. 4. Dek-FLAG co-immunoprecipitates the 70 kDa isoform of hDaxx, HDAC II, acetylated histone H4 and core histones. Fractions from the Sephacryl S-300 column that contained hDaxx, HDAC II, acetylated histone H4 and Dek-FLAG were pooled (fractions 9-13, 25ml total) and immunoprecipitated using an anti-FLAG affinity resin to isolate Dek-FLAG and associated proteins. After extensive washing, the proteins were eluted with a FLAG-specific peptide (5 ml final volume). (A) Silver stain analysis of bound proteins. Bound proteins from lysates not expressing Dek-FLAG (left lane) and lysates expressing Dek-FLAG (right lane) were separated by 4-20% SDS-PAGE and visualized by silver staining. (B) Western blot analysis of bound proteins. Equal volumes of the bound (left lane) and unbound (right lane) fractions were separated by 4-20% SDS-PAGE, and the proteins were detected with a western blot analysis using anti-hDaxx, anti-HDAC II or anti-acetylated histone H4 antibodies.