SMAD specific E3 ubiquitin protein ligase 1 accelerates diabetic macular edema progression by WNT inhibitory factor 1

Figure 5 Insulin like growth factor 2 mRNA binding protein 2 upregulated SMAD-specific E3 ubiquitin protein ligase 1 expression in an N6-methyladenosine modification-dependent manner. A: The N6-methyladenosine (m6A) modification site of SMAD-specific E3 ubiquitin protein ligase (SMURF) 1 was predicted by the SRAMP database; B: ARPE-19 cells were treated with normal concentration of glucose (NG) or high concentration of glucose (HG), and the m6A level of SMURF1 was then detected by methylated RNA immunoprecipitation (RIP); C: ARPE-19 cells were treated with NG or HG, and then the expression of insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) was detected by western blot; D: The interaction between IGF2BP2 protein and SMURF1 mRNA was verified by RIP method; E: ARPE-19 cells were transfected with IGF2BP2 knockdown construct or vector, and then cells were treated with dactinomycin (10 µg/mL) for 0, 2, 4, or 6 hours, followed by quantitative real-time PCR (qRT-PCR) to detect the stability of SMURF1 mRNA; F: IGF2BP2 or vector knockdown ARPE-19 cells were collected, and then the mRNA levels of IGF2BP2 and SMURF1 were detected by qRT-PCR; G: ARPE-19 cells were transfected with IGF2BP2 knockdown construct or vector, followed by western blot to detect the protein levels of IGF2BP2 and SMURF1. The experiments were repeated three times. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. SMURF1: SMAD-specific E3 ubiquitin protein ligase 1; IGF2BP2: Insulin like growth factor 2 mRNA binding protein 2; HG: High concentration of glucose; DFX: Desferrioxamine mesylate; NG: Normal concentration of glucose.