Methylation of AE9a in vivo and in vitro. (A) In vivo methylation of AE9a. Lysates of 293T cells coexpressing HA-PRMT1 and FLAG-AE9a was subjected to immunoprecipitation by anti-aDMA followed by Western blot analysis with FLAG Ab. Control lane (C) represents 293T cells transfected with empty vector. (B) PRMT1 can methylate AE9a in vitro at R142 residue. FLAG-tagged AE9a, WT or R142A, was transiently transfected and expressed in 293T cells. Control lane (C) represents 293T cells transfected with empty vector. After transfection, cells were treated with 20μM Adox. Nuclear extracts (NEs) were made and subjected to FLAG immunoprecipitation to enrich AE9a. Bead-bound AE9a WT was incubated with recombinant GST-PRMT1 and ³H-SAM (for fluorography) or unlabeled SAM (for Western blot analysis) for in vitro methylation. Results were analyzed by SDS-PAGE, and methylated FLAG-AE9a WT was either visualized by fluorography or detected by Western blot analysis with methyl RUNT (R142) Ab. (C) Arg142 residue of AE9a is the main methylation site for PRMT1. After FLAG-IP enrichment, equal amount of WT or R142A AE9a was used to incubate with recombinant GST-PRMT1 and ³H-SAM for in vitro methylation. Results were analyzed by SDS-PAGE, and methylated FLAG-AE9a was visualized by fluorography. (D) Methylation on AE9a by PRMT1 is much weaker than histone H4. AE9a and various amounts of histone H4 were used in methyltransferase assay. Protein methylation was analyzed by SDS-PAGE and visualized by fluorography. Methylation on AE9a was compared with histone H4 to obtain relative methylation status.