Fig. 4

Prmt1 methylated Ncoa4 at the R242 residue. (A) Different regions of the Ncoa4 protein: R1 (1–150 aa), R2 (151–450 aa), and R3 (451–625 aa). (B) Prmt1 methylated the R2 region of Ncoa4 in vitro. An in vitro methylation assay was performed using recombinant Prmt1 and His-Ncoa4, His-Ncoa4^R1, His-Ncoa4^R2, and His-Ncoa4^R3. Immunoblots were conducted using anti-ADMA, anti-His, and anti-Prmt1 antibodies. (C) Prmt1 methylated the R242 residue of Ncoa4. An in vitro methylation assay was performed using recombinant Prmt1 and His-Ncoa4 or its mutants (Ncoa4^R182K, Ncoa4^R242K, Ncoa4^R342K, or Ncoa4^R366K). Immunoblots were conducted using anti-ADMA, anti-His, and anti-Prmt1 antibodies. (D) Prmt1 methylated the R242 residue of Ncoa4 in MC3T3-E1 cells. MC3T3-E1 cells were co-transfected with Flag-Prmt1 and HA-Ncoa4 or its mutants (Ncoa4^R182K, Ncoa4^R242K, Ncoa4^R342K, or Ncoa4^R366K). Forty-eight hours after transfection, the cells were lysed and immunoprecipitated with anti-Flag agarose, followed by immunoblot analyses using anti-Flag, anti-Ncoa4, and anti-ADMA antibodies