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Fig. 3 | Biology Direct

Fig. 3

From: Prmt1-mediated methylation regulates Ncoa4 stability to transactivate Adamts genes and promote bone extracellular matrix degradation in chronic hematogenous osteomyelitis

Fig. 3

Prmt1 methylated Ncoa4 and promoted the assembly of the Ncoa4-Cbp-Ap1 complex. (A) Total protein methylation levels in tibial tissue homogenates from Control and CHOM mice. Homogenates were prepared by mixing three independent tibiae of equal weight from the same groups of mice. Left panel: Coomassie Brilliant Blue (CBB) staining confirmed equal total protein loading. Right panel: Immunoblot analysis revealed global protein methylation levels. (B) Prmt1 interacted with Ncoa4, Cbp, and Ap1 subunits in vivo. Protein extracts from CHOM tibial tissues were immunoprecipitated using IgG and anti-Prmt1-conjugated protein G. Both input and immunoprecipitated samples were probed for Prmt1, Ncoa4, Cbp, c-Jun, and c-Fos. (C) Prmt1 interacted with Ncoa4 in vitro. MC3T3-E1 cells were co-transfected with Flag-Prmt1 and HA-Ncoa4. After 48 h, cells were lysed and immunoprecipitated using anti-Flag agarose. Input and immunoprecipitated samples were analyzed using anti-Flag, anti-HA, and anti-ADMA antibodies. (D–F) Determination of interactions between Prmt1 and Cbp (D), Prmt1 and c-Jun (E), and Prmt1 and c-Fos (F). (G) Ncoa4 directly interacted with Cbp but not with Ap1 subunits in Co-IP assays. (H) Cbp directly interacted with both Ncoa4 and Ap1 subunits in Co-IP assays. (I) Ncoa4 immunoprecipitated Cbp and Ap1 subunits in CHOM tibial tissues. (J) Ncoa4 immunoprecipitated Cbp and Ap1 subunits in IL-1β-treated MC3T3-E1 cells. (K) TCE-5003 treatment dose-dependently inhibited the assembly of the Ncoa4-Cbp-Ap1 complex

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