Ab412

Ten-thousand small-molecule compounds from a DIVER^Set library (ChemBridge) were screened as follows: High-binding 96-well plates (Fisher Scientific) were coated with 100 ng of recombinant SmD3 (the substrate) at 4 ^oC overnight. After blocking with 5% BSA for 30 min at room-temperature, PRMT5-WDR77 (800 ng) was added to each well. DIVER^Set compounds were then transferred to individual wells to a final concentration of 20 μM. The enzyme reaction was initiated by the addition of the methyl donor (AdoMet, 10 μM). Reaction mixtures were incubated for 3 h at 30 ^oC. The wells were then washed twice with TBST (25 mM Tris [pH 7.5], 150 mM NaCl, 0.1% Tween 20) and blocked in TBST containing 2% BSA for 30 min. The methylarginine-specific primary antibody ab412 (Abcam) (1:1,000) and secondary antibody (anti-mouse IgG-peroxidase at 1:10,000; Fisher Scientific) were added to each well and the mixtures were incubated at room-temperature for 1 h. After three washes with TBST, 0.1 ml of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (0.1 mg/ml) was added to each well. Wells were incubated for 5 min in the dark at room-temperature and the reaction was stopped by addition of 0.1 ml of 2 M H₂SO₄ per well. Potential “hits” were compounds associated with at least a 90% reduction in optical density at 450 nm (in duplicate).