a, HA tagged full length L(3)MBT (HA-MBT) was expressed in S2 cells and monitored after RNAi against l(3)mbt. b, PR-Set7 levels in PR-Set7 and l(3)mbt knockdown S2 cells. c, Blots of extracts from S2 cells treated for five days with RNAi, probed with anti-mono Histone H4K20 (mono), anti-histone H3 (H3) and anti-Set7 antibodies (Set7). The graph shows the average ratio of each value to the values of lacZ cells. The value of monomethylated H4K20 was normalized to the value of histone H3. d, De novo methylation was assessed by supplying tritium-labeled SAM, to S2 cells treated with different RNAis. The graph shows the average ratio of de novo histone H4 methylation to total de novo histone methylation of H2A, H2B and H3. Error bars for both graphs show two SDs (n = 3). The knockdown of l(3)mbt does not affect the level of tritium labeling on histone H4, showing that newly monomehtylated H4K20 is stable for duration of the experiment even in the absence of L(3)MBT.