At the protein level, bands of 59 kD and 49 kD, correspond ing to HDACs 2 and 3, respectively, appeared to be most abundant. This is similar to a previous report of the pat tern of HDACs selleckchem Sorafenib expressed in normal brain tissue. To examine the change in HDAC expression in response to ONC, quantitative RT PCR analysis was con ducted on samples isolated from several time points fol lowing surgery. By 1 day post ONC, the mRNAs for Hdacs 2 and 3 doubled, while transcripts for Hdacs 1 and 5 showed modest, but not significant increases. At 3 days post ONC, only Hdac3 transcripts remained significantly elevated. By 7 days post ONC, no significant elevation of any of the examined Hdacs was detected.

HDACs 2 and 3 in injured retinas Since both Hdac 2 and 3 transcripts showed a significant increase following ONC and the majority of HDAC activ ity was sensitive to apicidin, we characterized these HDACs in the retina before and after ONC. To determine the localization of these isoforms, immunofluorescence was performed on retinal cryosections from control and crush eyes. Both HDAC2 and HDAC3 were present in cells of the ganglion cell layer and inner nuclear layer of control eyes. At higher magnifi cation of the GCL of control retinas, HDAC2 colocalized with 4,6 diamindino 2 phenylindole, indicating that it was nuclear, con sistent with reports that this is an exclusively nuclear pro tein. In contrast, HDAC3 labeling was diffuse and appeared to be predominantly cytoplasmic with minimal overlap with DAPI labeling.

This was also in agreement with previous reports that HDAC3 contains both a nuclear localization signal and a nuclear export signal, and can exist in both the cytoplasm and nuclei of cells. Because subcellular localization of HDACs is a com mon mechanism of controlling HDAC activity, we also examined the distribution of HDAC2 and HDAC3 after ONC. In sections from retinas 3 days post ONC, HDAC2 remained localized to nuclei of the GCL. HDAC3 localization in the GCL, how ever, appeared to change after ONC, with cells showing both cytoplasmic and nuclear localization, as determined by colabeling with DAPI staining. To confirm these findings, west ern blot analysis was conducted on nuclear and cytoplas mic fractions isolated from control and crush retinas. As shown in Figure 3C, a band at 59 kD, corresponding to HDAC2, was present in the nuclear fractions of both con trol and ONC retinas.

A 49 kD band, corresponding to HDAC3, was present in the cytoplasmic fraction Anacetrapib from control retinas, but was both cytoplasmic and nuclear in the experimental crush retinas consistent with the nuclear translocation of this protein after ONC. As a con trol for the fractionation, the blots were also probed for acetylated histone H4 and GAPDH as nuclear and cyto plasmic controls, respectively. Histone H4 acetylation in the GCL decreases following ONC Histones H3 and H4 are substrates of HDACs 2 and 3.