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. 2017 Aug 30;45(20):11700–11710. doi: 10.1093/nar/gkx775

Figure 5.

Figure 5.

PP32 knock-down affects the maturation of newly synthesized histone H4. (A) Western blot of HA-Hsp90 pull-down assay from cytosolic extracts derived from siControl and siHsp90 treated HeLa cells. (B) Graph of the tryptophan fluorescence spectroscopy of Hsp90 upon titrating full length histone H4 (left), or unmodified or acetylated amino acids 1–20 of histone H4 (right). ΔF is the difference between initial fluorescence (F0) and the fluorescence after ligand addition (FQ). KD is the dissociation constant of the ligand with Hsp90. (C) Left, Western blot analysis of thermal stability assay using HeLa S100 extracts. 20 μg of S100 extracts were heated for 20 min at the indicated temperatures and centrifuged at 10 000 × g for 5 min. Supernatant and pellet were analyzed by western blot. Right, graph of the percentage of either total or acetylated histone H4 found on the pellet, taken as 100% of the sum of the supernatant and pellet at each temperature. Standard deviations were taken from three independent experiments. *P < 0.05, Student′s t-test. (D) Thermal stability of S100 extracts derived from siControl and siPP32 HeLa cells, as described in (C). (E) Thermal stability of S100 extracts derived from siControl and siPP32 HeLa cells, as described in (C), in the absence or presence of recombinant PP32.