Ab101661

This assay was conducted by Shanghai Huawei pharmaceutical Co. Ltd., China. HDAC 1 and 6 enzymes were purchased from Abcam (nos. AB101661 and AB42632). HDAC 2, 3, 4, 8, and 11 enzymes were purchased from SignalChem (nos. H84-30G, H85-30G, H86-31G, H90-30H, and H93-30G). All of the enzymatic reactions were conducted at 37 °C for 30 min. The 50 µL reaction mixture contains 25 mM Tris, pH 8.0, 1 mM MgCl₂, 0.1 mg/mL BSA, 137 mM NaCl, 2.7 mM KCl, HDAC, and the corresponding enzyme substrate (the enzyme substrate of HDAC 1, 2, 3, and 6 is Ac-Leu-GlyLys(Ac)-AMC; the enzyme substrate of HDAC 4, 5, 7, 8, and 9 is Ac-Leu-Gly-Lys(Tfa)-AMC; the enzyme substrate of HDAC 10 is Ac-Arg-His-Lys(Ac)-Lys(Ac)-AMC). The compounds were diluted in 10% DMSO, and 5 µL of the dilution was added to a 50 µL reaction so that the final concentration of DMSO is 1% in all of reactions. The assay was performed by quantitating the amount of fluorescent product in solution following an enzymatic reaction. Fluorescence is then analyzed with excitation at 350–360 nm and emission at 450–460 nm using a SpectraMax M5 microtiter plate reader. The IC₅₀ values were calculated using nonlinear regression with normalized dose-response fit using GraphPad Prism software.

HDAC1 and HDAC6 were purchased from Abcam (AB101661 and AB42632), HDAC2 and HDAC3 were purchased from SignalChem (H84-30G and H85-30G). HDACs substrate Boc-Lys(acetyl)-AMC was purchased from Bachem. Pan-HDACs inhibitor SAHA, HDAC6 selective inhibitor tubastatin A and proteasome inhibitor carbobenzoxy-Leu-Leu-leucinal (MG132) were purchased from Selleckchem. The following antibodies were used for western blot: acetylated α-tubulin antibody (Sigma: T6793, 1:1000), β-actin antibody (Sigma: A1978, 1:5000), acetylated histone H4 antibody (Abcam: ab15823, 1:1000), anti-mouse IgG HRP conjugate (Promega: W402B, 1:10000) and anti-rabbit IgG HRP conjugate (Promega: W401B, 1:10000). The following antibodies were used for immunofluorescent staining: HDAC6 antibody (Santa Cruz: sc-11420, 1:200), ubiquitin antibody (Millipore: 05-1307, 1:500) and Alexa Fluor^® 647 goat anti-rabbit IgG (Invitrogen: A21245, 1:200).