Journal: Investigative Ophthalmology & Visual Science

Article Title: Changes in Class I and IIb HDACs by δ-Opioid in Chronic Rat Glaucoma Model

doi: 10.1167/iovs.61.14.4

Figure Lengend Snippet: 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.

Article Snippet: The enzyme activity of HDACs was measured using the 4-methylcoumarin-7-amide (AMC), a fluorophore conjugated to acetylated lysine substrates, Boc-Lys(Ac)-AMC (Cat # K-6405, Aurum Pharmatech) or (Boc-Lys(Tfa)-AMC (Cat # I-1985, Bachem), as described previously., Retina extract was prepared using lysis buffer (50 mM Tris-base, 10 mM EDTA, 0.5 mM sodium orthovanadate, 0.5% sodium deoxycholic acid, 1% Triton X-100, and cocktail protease inhibitor).

Techniques: Activity Assay, Incubation