Figure 2.
PRMT1 methylates FLT3 at R972 and R973. (A) Green fluorescent protein (GFP)–tagged type I PRMTs were expressed in 293T cells (ectopically expressing FLT3-WT), and then total cell lysates were pulled down with anti-FLT3 antibody, followed by western blotting of FLT3 and GFP. (B) Western blot indicating PRMT1 and FLT3 interaction in SEM cells based on immunoprecipitation (IP) with anti-rabbit immunoglobulin G (IgG) or anti-FLT3 antibodies, followed by western blotting of FLT3 and PRMT1. (C) Representative images from an in situ proximity ligation assay of primary MLL-r ALL cells. Top row (left): red fluorescent spots indicate PRMT1/FLT3 protein interaction, (middle) DAPI-stained nuclei are blue, and (right) merged image. Scale bar, 10 μm. Bottom row: IgG controls. (D) Left: HA-tagged FLT3 fragments and GFP-tagged PRMT1 were co-expressed in 293T cells, and indicated FLT3 protein fragments were pulled down with anti-HA antibody, followed by (middle) western blotting for HA and GFP. Right: quantitative intensity analysis of interaction of FLT3 domains with PRMT1. (E) Western blotting for pan-ADMA levels in MOCK-, FLT3-WT-, FLT3-R655K-, FLT3-R707K-, FLT3-R972K-, or FLT3-R973K-transduced 293T cells. Respective lysates were pulled down with anti-FLT3 antibody. (F) Endogenous FLT3 protein was pulled down from SEM cells followed by MS analysis. R972/973 was identified as dimethylated (Di). The mass-to-charge ratio (m/z) of "y7-y4" indicated the total molecular weight of 3 amino acid residues including N residue and 2 R residues (2Me). (G) Amino acid sequence of peptides corresponding to the FLT3 966-980 region, in which R972/973 was modified as indicated. Different doses of peptides were detected by anti-FLT3 966-980 (control) or anti-FLT3 R972/973me2a antibodies. (H) Western blot of FLT3, R972/973me2a, or PRMT1 in SEM cells transduced with inducible shPRMT1. (I) In vitro methylation assay of indicated GST-tagged FLT3 peptides (aa841-aa993) with or without PRMT1 enzyme plus S-adenosyl methionine (SAM). (J) MS analysis of FLT3 peptide (left) without or (right) with PRMT1 enzyme plus SAM. HA, human influenza hemagglutinin; m/z,  represents mass divided by charge number and the horizontal axis in a mass spectrum is expressed in units of m/z.

PRMT1 methylates FLT3 at R972 and R973. (A) Green fluorescent protein (GFP)–tagged type I PRMTs were expressed in 293T cells (ectopically expressing FLT3-WT), and then total cell lysates were pulled down with anti-FLT3 antibody, followed by western blotting of FLT3 and GFP. (B) Western blot indicating PRMT1 and FLT3 interaction in SEM cells based on immunoprecipitation (IP) with anti-rabbit immunoglobulin G (IgG) or anti-FLT3 antibodies, followed by western blotting of FLT3 and PRMT1. (C) Representative images from an in situ proximity ligation assay of primary MLL-r ALL cells. Top row (left): red fluorescent spots indicate PRMT1/FLT3 protein interaction, (middle) DAPI-stained nuclei are blue, and (right) merged image. Scale bar, 10 μm. Bottom row: IgG controls. (D) Left: HA-tagged FLT3 fragments and GFP-tagged PRMT1 were co-expressed in 293T cells, and indicated FLT3 protein fragments were pulled down with anti-HA antibody, followed by (middle) western blotting for HA and GFP. Right: quantitative intensity analysis of interaction of FLT3 domains with PRMT1. (E) Western blotting for pan-ADMA levels in MOCK-, FLT3-WT-, FLT3-R655K-, FLT3-R707K-, FLT3-R972K-, or FLT3-R973K-transduced 293T cells. Respective lysates were pulled down with anti-FLT3 antibody. (F) Endogenous FLT3 protein was pulled down from SEM cells followed by MS analysis. R972/973 was identified as dimethylated (Di). The mass-to-charge ratio (m/z) of "y7-y4" indicated the total molecular weight of 3 amino acid residues including N residue and 2 R residues (2Me). (G) Amino acid sequence of peptides corresponding to the FLT3 966-980 region, in which R972/973 was modified as indicated. Different doses of peptides were detected by anti-FLT3 966-980 (control) or anti-FLT3 R972/973me2a antibodies. (H) Western blot of FLT3, R972/973me2a, or PRMT1 in SEM cells transduced with inducible shPRMT1. (I) In vitro methylation assay of indicated GST-tagged FLT3 peptides (aa841-aa993) with or without PRMT1 enzyme plus S-adenosyl methionine (SAM). (J) MS analysis of FLT3 peptide (left) without or (right) with PRMT1 enzyme plus SAM. HA, human influenza hemagglutinin; m/z,  represents mass divided by charge number and the horizontal axis in a mass spectrum is expressed in units of m/z.

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