Fig. 2

AML1-ETO is methylated at Lys43 in vitro and in vivo. a Western blotting for anti-ETO immunoprecipitates (lanes 1, 2) from Kasumi-1 and SKNO-1 cells. IgG pull-down performed in parallel (lanes 3, 4) was used as a negative control. b Kasumi-1 and SKNO-1 cells were transfected with AML1-ETO shRNA (shAE) for 48 h. The anti-ETO immunoprecipitates (IP) and total cell lysates (Input) were subjected to Western blotting. c Western blotting for anti-ETO immunoprecipitates (lanes 1, 2) from AML1-ETO-positive patient primary cells (N = 2). IgG pull-down performed in parallel (lanes 3, 4) was used as a negative control. Pt patient. d Upper: Diagram of AML1-ETO domain constructs; lower: Western blotting for anti-His immunoprecipitates from HEK293 cells expressing His-AE-W (wild type) (lane 1), His-AE-ΔNHR1 (lane 2), His-AE-ΔNHR2 (lane 3) or His-AE-ΔRUNT (lane 4). e The MS/MS fragmentation spectrum and pattern of the isolated peptide MSEALPLGAPDAG AALAGK. AML1-ETO was immunoprecipitated by anti-ETO from Kasumi-1 cells. (Inset) SDS-PAGE of precipitated proteins confirmed AML1-ETO pulldown (Coomassie blue-stained) in Kasumi-1 (lane 2) and HEK293 cells (lane 3). Lane 1 is the protein ladder marker. The AML1-ETO bands from the anti-ETO IP (indicated by star) were subjected to LC/MS/MS. The b-type and y-type product ions are marked in the spectrum. A 14 Da increase in the Lys43 residue was observed, which indicated a mono-methylated lysine residue. f Western blotting for anti-HA immunoprecipitates from HEK293 cells expressing HA-AE-W (lane 2), HA-AEK24R (lane 3), or HA-AEK43R (lane 4). IP Immunoprecipitation, WB Western blotting; AE AML1-ETO. The “Input” of each Fig is the immunoblot analysis for whole cell extracts to show the target protein levels. meK43: our anti-AML1-ETO Lys43 antibody; meK: commercial lysine methylation antibody. The data are representative of 3 independent experiments. Source data are included in the Source Data file