Fig. 1

Quality control of the anti-H3K4ox antibody. a Schematic representation of the LOXL2 reaction. The red circle indicates the intermediate residue that is targeted by the in-house generated anti-H3K4ox antibody. b The artificial amino acid 6-hydroxynorleucine was used in the peptide to generate the anti-H3K4ox antibody. c The anti-H3K4ox antibody was found to be specific in western blot in two replicates of dot blots of dilution series of oxidized histone H3 peptide (H3K4ox) or unmodified H3 peptide (left panel), as well as in a representative dot blot of a dilution series of H3K9me3, H3K4ox, H3K4me3, or H3 peptides (right panel). d Nucleosomes were incubated with recombinant wild-type (wt) LOXL2 or a catalytically inactive LOXL2 (mut) purified from baculovirus to detect H3K4ox/H3K4me3 levels (upper panel). Lysates of MCF-7 cells transfected with an empty vector (ø) or with LOXL2 were analyzed by western blotting, using the indicated antibodies (lower panel). e Western blot for LOXL2, H3K4ox, and total H3 from MDA-MB-231 cells infected with short hairpin RNA (shRNA) as a control, or a knockdown (KD) using a shRNA specific for LOXL2 (LOXL2 KD) (left panel). Anti-H3K4ox ChIP-PCR was used to analyze the E-cadherin gene (CDH1) promoter in MDA-MB-231 cells infected with shRNA for either control (green bar) or LOXL2 KD (blue bar). Data of qPCR amplifications were normalized to the input and to total H3 for each condition. Error bars indicate the SD from at least three independent experiments. **P < 0.01. f Western blot of H3K4ox and biotin incorporation (BTH-H3) in nucleosomes incubated with recombinant LOXL2 purified from baculovirus, after different incubation times