American Society of Hematology

Figure 5.

$Figure 5. HDAC inhibitors induce acetylation of different intracellular targets. (A) Monocytes stimulated for 3 hours with LPS in the absence or presence of ITF2357 (20 nM or 100 nM), TSA (1 μM), HC-toxin (HC-T; 1 μM), SAHA (0.5 μM), or tubacin (20 μM) were fractionated to obtain nuclei, which were subjected to SDS-PAGE and Western blotting. Filters were hybridized with anti-histone H4 (bottom panel) or anti-acetyl-H4 (top panel). (B) Monocytes stimulated for 3 hours with LPS in the absence or presence of SAHA (0.5 μM), ITF2357 (0.1 μM), HC-toxin (HC-T; 1 μM), or tubacin (tuba; 20 μM) were lysed and cell lysates were analyzed by Western blotting with anti-HSP90 (top panel) or anti-acetyl-lysine (bottom panel). The arrow in the top panel indicates HSP90; the arrow in the bottom panel indicates a 50-kDa band, probably corresponding to acetylated tubulin-α. (C) Monocytes treated as in panel A were fixed, permeabilized, and stained with anti-tubulin-α (not shown) or anti-acetyl-tubulin-α and analyzed by cytofluorimetry. Monocytes treated with the different inhibitors stained equally with anti-tubulin-α (not shown) but differently with anti-acetyl-tubulin-α. Data are expressed as mean fluorescence intensity (mean FI) of cells stained with anti-acetyl-tubulin-α. (D) Monocytes treated as in panel B were lysed and analyzed by Western blotting with anti-tubulin-α (top panel) or anti-acetyl-tubulin-α (bottom panel). Arrows indicate tubulin-α and acetyl-tubulin-α, respectively. For each panel, one representative experiment of at least 4 performed is shown.$

HDAC inhibitors induce acetylation of different intracellular targets. (A) Monocytes stimulated for 3 hours with LPS in the absence or presence of ITF2357 (20 nM or 100 nM), TSA (1 μM), HC-toxin (HC-T; 1 μM), SAHA (0.5 μM), or tubacin (20 μM) were fractionated to obtain nuclei, which were subjected to SDS-PAGE and Western blotting. Filters were hybridized with anti-histone H4 (bottom panel) or anti-acetyl-H4 (top panel). (B) Monocytes stimulated for 3 hours with LPS in the absence or presence of SAHA (0.5 μM), ITF2357 (0.1 μM), HC-toxin (HC-T; 1 μM), or tubacin (tuba; 20 μM) were lysed and cell lysates were analyzed by Western blotting with anti-HSP90 (top panel) or anti-acetyl-lysine (bottom panel). The arrow in the top panel indicates HSP90; the arrow in the bottom panel indicates a 50-kDa band, probably corresponding to acetylated tubulin-α. (C) Monocytes treated as in panel A were fixed, permeabilized, and stained with anti-tubulin-α (not shown) or anti-acetyl-tubulin-α and analyzed by cytofluorimetry. Monocytes treated with the different inhibitors stained equally with anti-tubulin-α (not shown) but differently with anti-acetyl-tubulin-α. Data are expressed as mean fluorescence intensity (mean FI) of cells stained with anti-acetyl-tubulin-α. (D) Monocytes treated as in panel B were lysed and analyzed by Western blotting with anti-tubulin-α (top panel) or anti-acetyl-tubulin-α (bottom panel). Arrows indicate tubulin-α and acetyl-tubulin-α, respectively. For each panel, one representative experiment of at least 4 performed is shown.

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