DAXX down-regulation generates a soluble pool of H3.3. (A) FRAP analysis of H3.3-EGFP incorporated into chromatin in DAXX- and sham (H2O)–depleted cells 96 h after transfection (mean ± SD of five cells). (B) Western blot analysis of H3.3-EGFP distribution in DAXX- and sham (control)–depleted cells. Cell lysates (Inp, input) were fractionated into 1% Triton X-100 soluble (sol) and insoluble (Ins) fractions. H3.3-EGFP is detected with anti-GFP antibodies. H3 is shown as a chromatin marker. ASF1A is shown as a marker of the soluble fraction. (C) Redistribution of H3.3-EGFP after siRNA-mediated knockdown of PML. (D) Western blot analysis of H3.3-EGFP distribution in Triton X-100 soluble and insoluble fractions, from PML- and sham (control)–depleted cells. Cell lysates were fractionated as in B.
