Fig. 5

Rnf8 ubiquitinated Ncoa4 at the K49 residue. (A) Effects of IL-1β and MG132 on Ncoa4 methylation and ubiquitination in Prmt1^KD cells. Prmt1^KD cells were treated with IL-1β alone or co-treated with IL-1β and MG132 for 6 h, followed by an IP assay using anti-Ncoa4 agarose. The purified proteins were probed with anti-Ncoa4, anti-ADMA, and anti-Ubiquitin antibodies. (B) Effects of IL-1β, TCE-5003 (or MS023), and MG132 on Ncoa4 methylation and ubiquitination in MC3T3-E1 cells. MC3T3-E1 cells were treated with IL-1β alone, co-treated with IL-1β and TCE-5003 (or MS023), or co-treated with IL-1β, TCE-5003 (or MS023), and MG132 for 6 h, followed by an IP assay using anti-Ncoa4 agarose. The purified proteins were probed with anti-Ncoa4, anti-ADMA, and anti-Ubiquitin antibodies. (C) Ncoa4 underwent arginine methylation, but not ubiquitination, in CHOM tibial tissues. Tibial tissues from control and CHOM mice (n = 3 per group) were subjected to IP assay using anti-Ncoa4-conjugated agarose beads. The IP products were analyzed by immunoblotting with anti-Ncoa4, anti-ADMA, and anti-Ubiquitin antibodies. (D and E) Rnf8 interacted with Ncoa4 in Prmt1^KD cells. The Prmt1^KD cells were lysed and immunoprecipitated using anti-Ncoa4 agarose (D) or anti-Rnf8 agarose (E). The purified proteins were probed with anti-Rnf8 and anti-Ncoa4 antibodies. (F) Rnf8 ubiquitinated the Ncoa4^R1 region in vitro. An in vitro ubiquitination assay was performed using E1, E2, recombinant GST-Rnf8, and His-Ncoa4 or its truncated mutants (Ncoa4^R1, Ncoa4^R2, and Ncoa4^R3). Immunoblots were conducted using anti-GST, anti-His, and anti-Ubiquitin antibodies. (G) Rnf8 ubiquitinated Ncoa4 at the K49 residue in vitro. An in vitro ubiquitination assay was performed using recombinant GST-Rnf8 and His-Ncoa4 or its mutants (Ncoa4^K42A, Ncoa4^K49A, Ncoa4^K81A, Ncoa4^K112A, or Ncoa4^K131A). Immunoblots were conducted using anti-GST, anti-His, and anti-Ubiquitin antibodies. (H) RNF8 ubiquitinated Ncoa4 at the K49 residue in MC3T3-E1 cells. MC3T3-E1 cells were co-transfected with Flag-Rnf8 and HA-Ncoa4 or its mutants (Ncoa4^K42A, Ncoa4^K49A, Ncoa4^K81A, Ncoa4^K112A, or Ncoa4^K131A). Forty-eight hours after transfection, the cells were lysed and immunoprecipitated with anti-Ncoa4 agarose, followed by immunoblot analyses using anti-Flag, anti-HA, and anti-Ubiquitin antibodies