Figure 7.

Ezh2 regulates TRAF6 degradation and its downstream signaling. (A and B) Analysis of TRAF6 Lys48-linked ubiquitination in WT and Ezh2-deficient macrophages (A) or in Ezh2-KO BV2 cell that infected with EV or lentiviral vector encoding WT Ezh2 (B) that was stimulated with LPS at the indicated time points. (C–E) IB analysis of TRAF6 protein levels in WT and Ezh2-deficient macrophages (C), in microglia (D), or in Ezh2-KO BV2 cells that were infected with EV or lentiviral vector encoding WT Ezh2 (E) that was stimulated without or with LPS at the indicated time points. The relative expression of Traf6 compared with loading controls (Actin or Hsp60) was quantified and presented below the Traf6 IB panels. (F and G) qRT-PCR analysis of Traf6 mRNA expression in WT and Ezh2-deficient macrophages (F) or microglia (G) that was stimulated with LPS at the indicated time points. (H–K) Immunoblot analysis of phosphorylated (P-) and total NF-κB and MAPKs signaling proteins, H3K27me3, H3, Ezh2, or Hsp60 (loading control) in whole-cell lysates of WT and Ezh2-deficient macrophages that were left unstimulated or stimulated with LPS (100 ng/ml; H) or Pam3CSK4 (100 ng/ml; I) or poly I:C (pI:C, 20 µg/ml; J), and IB of the indicated signal protein in the whole-cell lysates of WT and Ezh2-deficient primary microglia that were left unstimulated or stimulated with LPS (100 ng/ml; K) at the indicated time points. The qRT-PCR data were normalized to a reference gene, Actb (β-actin), and other data are shown as mean ± SD based on three independent experiments.