Research Paper Volume 12, Issue 4 pp 3126—3139

Figure 3. AK045171 binds to SP1 and regulates the expression of MG53. (A) RNA pull-down and silver staining assays were performed to investigate the potential proteins that combined with AK045171. (B) RNA pull-down and (C) RIP assays were used to confirm the interaction between AK045171 and SP1. (D) We next evaluated the location of AK045171 in cardiomyocytes and found that AK045171 primarily exists in the cell nucleus. (E, F). Immunofluorescence was used to further detect the expression and location of AK045171. (G) The entire sequence of AK045171 was divided into 4 sections to determine which region binds with SP1 using an RNA pull-down assay. (H, I) Luciferase activity assays were performed to detect whether knockdown or overexpression of AK045171 influences SP1 transcription. (J) ChIP assay was used to determine the binding of SP1 and the promoter of MG53 after AK045171 knock down. (K) Western blot was used to detect the expression of MG53 under AK045171 overexpression or knock down. (L) Evaluation of the MG53 protein level after SP1 deletion with a Crispr/cas9 system. *p<0.05 vs the IgG, si-control, Ad-vector or wt group; ^# p<0.05 vs Ad-vector group.