Figure 2.

Measurement of histone deacetylases (HDACs) activity (a), and effects of SNC-121 on HDAC activity (b) in retina extracts obtained from normal Brown Norway rats. Enzyme activity for selective HDAC was measured using specific conjugated fluorophore acetylated lysine substrates, Boc-Lys(Ac)-AMC or Boc-Lys(Tfa)-AMC for HDAC 1, 2, 3, and 6 or HDAC 4, 5, 7 to 11, respectively, as shown in a. To determine the specificity of assay, equal amount of retina extract (1 µg) was pre-incubated with vehicle (DMSO) or 1µM Trichostatin A (TSA) for 15 minutes before adding specific substrates as shown in a. Additionally, we measured the effects of SNC-121 treatment on HDAC 1, 2, 3, and 6 activity using Boc-Lys(Ac)-AMC as substrate in normal animals as shown in b. Normal Brown Norway rats were treated with 1 mg/kg SNC-121 (i.p.) once a day for 7 days. On day 7, animals were euthanized and retinas were collected and analyzed for HDAC class I and IIb enzyme activity. Equal amount of retina extract (1 µg) was incubated with specific substrate. After 1 hour, trypsin was added and the HDAC activity was quantified by detection of released fluorescent amino-methoxy cumarin (Ex/Em at 355/460). Data are expressed as mean ± SE. ****P < 0.0001; n = 6.