Figure 6. Compound 3195 inhibits JARID1B H3K4 demethylase activity.

(A) 5 µg of purified calf thymus histones were incubated with 240 ng of purified JARID1B, in the presence of 1 mM α-KG, 0.1 mM (NH₄)₂Fe(SO₄)₂ and 2 mM Ascorbate. 2,4-PDCA (PDCA), 3195 compound (3195) or DMSO were added as indicated. After 3 h of incubation samples were run on a 15% SDS PAGE gel for western blot analysis. The filter was hybridized with antibodies against H3K4me3. Histone levels are based on coomassie blue staining. The Ponceau Red stained filter is also shown (PR). (B) Quantitation of the relative H3K4me3 demethylation upon incubation of JARID1B and the indicated concentrations of 3195. Data are adjusted to histone levels and normalized to the DMSO control, arbitrarily set as 1. Data are the average of three independent experiments. Standard deviation is indicated.