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. 2015 Oct 9;6(34):35173–35182. doi: 10.18632/oncotarget.6050

Figure 2. In vivo methylation of INCENP by PRMT1.

Figure 2

A. Determination of the titer and specificity of the anti-mono-methylated R887 INCENP antibody analyzed by ELISA. B. Validation of the anti-mono-methylated R887 INCENP antibody. Recombinant GST-tagged INCENP protein and S-adenosyl-L-methionine (SAM) were incubated in the presence of BSA or recombinant PRMT1. Samples were immunoblotted with the anti-mono-methylated R887 INCENP antibody, and amounts of loading proteins were evaluated by staining with MemCodeTM Reversible Protein Stain. C. 293T cells were co-transfected with a FLAG-INCENP-WT (amino acids 821-918) vector or a FLAG-INCENP-R887A (amino acids 821-918) vector and an HA-PRMT1 vector. The samples were immunoblotted with anti-mono-methylated R887 INCENP, anti-FLAG and anti-AURKB antibodies after immunoprecipitating with anti-FLAG M2 agarose (Sigma-Aldrich). D. HeLa cells were transfected with FLAG-PRMT1-WT and immunocytochemistry was performed with anti-FLAG (Sigma-Aldrich, M2, dilution: 1:100, green) and anti-R887meINCENP (dilution: 1:100, green) antibodies. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Transfected HeLa cells exhibited higher INCENP and R887meINCENP integrated densities.