Supplementary Figure 2: EHMT2 methylates KDM1A at K114 in vivo.
(a-d) Characterization of the rabbit polyclonal anti-KDM1A K114me2 specific antibody. (a,b) Anti-KDM1A K114me2 Western blots and Ponceau S stainings. Samples are peptides. (c,d) Anti-KDM1A K114me2 (c,d), anti-KDM1A and anti-tubulin (d) Western blots. Samples are nuclear extract from LNCaP cells (c), or extracts from LNCaP cells treated with siRNA control (Ctrl) or siRNA against KDM1A (d). (e) Anti-KDM1A K114me2, anti-KDM1A and anti-EHMT2 Western blots. Samples are recombinantly expressed and purified GST-KDM1A or GST-KDM1A K114A proteins that were incubated in the presence or absence of purified GST-EHMT2 aa786-1210 with or without SAM. (f, g) Anti-Flag, anti-KDM1A K114me2 and anti-KDM1A Western blots. Samples are extracts from 293T cells transfected with Flag-KDM1A or Flag-KDM1A K114A in the presence or absence of Flag-EHMT2 (f, g) and cultured with or without the EHMT2 inhibitor UNC0638 (g). Extracts were immunoprecipitated with anti-Flag antibody. (h) Coomassie blue stainings and anti- EHMT2 and anti-KDM1A K114me2 Western blots. Samples are expressed purified GST-KDM1A or GST-KDM1A K114A expressed alone or in combination with GST-EHMT2. (i) Demethylase activity assay. Samples are H3K4me2 peptides that were incubated with GST-KDM1A or GST-KDM1A K114A expressed alone or in combination with GST-EHMT2. (j) Anti-GST, anti-Strep and anti KDM1A Western blots. Samples are GST-PHF21A expressed either alone or in combination with Strep-RCOR1, His-KDM1A, or His-KDM1A aa171-852 and purified over Glutathione SepharoseTM 4B beads. (k) Anti-KDM1A Western blot. Samples are extracts from LNCaP cells that were immunoprecipitated with anti-KDM1A K114me2 antibody. Numbers represent the quantified KDM1A levels detected with the anti-KDM1A antibody. FT: flow-through; IP: immunoprecipitation. Results are representatives of 2 (a-d,g-k) and 4 (e,f) independent experiments.
