Figure 5.

The P62 Promotes the Methylation on IGFII mRNA and Its Expression in the TNF-α-Treated Mesenchymal Stem Cells Transfected with pCMV6-AC-GFP (GFP-Control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi Control), or pGFP-V-RS-P62 (P62 RNAi)

(A) RIP with anti-N6Ame followed by RT-PCR with IFGII mRNA primers. IgG RIP as negative control is shown. IGFII mRNA was used as INPUT. (B) Super-EMSA (gel-shift) with biotin-IGFII cRNA probe and anti-N6Ame antibody. The intensity of the band was examined by western blotting with anti-biotin. (C) (a) IGFII rRNA biotin-probe pull-down followed run on assay with anti-N6Ame. Biotin as internal control is shown. (b) IGFII rRNA biotin-probe pull-down followed northern-western blotting assay with anti-N6Ame is shown. Histone as internal control is shown. (D) (a) Western blotting with anti-IGFII in the TNF-α-treated mesenchymal stem cells transfected with pCMV-mir (control), pCMV-miR122, pCMV6-AC-GFP-P62 (GFP-P62), or pCMV-miR122 plus pCMV6-AC-GFP-P62 (GFP-P62). β-actin was used as an internal control. (b) The IGFII 3′ UTR luciferase activity assay in the TNF-α-treated mesenchymal stem cells transfected with pCMV-mir, pCMV-miR122, pCMV6-AC-GFP-P62, and pCMV-miR122 plus pCMV6-AC-GFP-P62 is shown. (E) RT-PCR with IGFII mRNA primers in the TNF-α-treated mesenchymal stem cells transfected with pCMV6-AC-GFP (GFP-control), pCMV6-AC-GFP-P62 (GFP-P62), pGFP-V-RS (RNAi control), or pGFP-V-RS-P62 (P62 RNAi). β-actin served as internal control. (F) Western blotting with anti-IGFII and anti-pIGFII in the TNF-α-treated mesenchymal stem cells transfected with pCMV6-AC-GFP, pCMV6-AC-GFP-P62, pGFP-V-RS, and pGFP-V-RS-P62. β-actin was used as an internal control. **p < 0.01.