Fig. 5

Circ_005077 bound to the CyPA protein and decreased its degradation via the ubiquitin-mediated proteasomal pathway. (A) The gel was stained with Coomassie bright blue to show the total proteins loading of ChIRP by circ_005077 probes and NC probes. (B)Venn diagram showed the differential proteins of LC-MS analysis. (C) H9c2 cells were stained with immunofluorescence to detect the colocalization of the CyPA protein (red, 400× magnification) and circ_005077 (green, 400× magnification). (D) The binding of circ_005077 to the CyPA protein was validated by ChIRP assay followed by Western blotting. (E) The CyPA protein was pulled down by RIP assay. (F) Enrichment of circ_005077 pulled down by the CyPA protein was shown. (G) The relative levels of CyPA mRNA in H9c2 cells with inhibition or overexpression of circ_005077 were shown. (H) CyPA protein levels were shown in H9c2 cells with inhibition or overexpression of circ_005077. (I) The expression of CyPA in H9c2 cells after inhibition or overexpression of circ_005077 and then treated with or without MG132 for 12 h, demonstrated by Western blotting. (J) The expression of CyPA was evaluated at different durations of CHX administration with inhibition or overexpression of circ_005077 in H9c2 cells. (K) Quantification of CyPA degradation rate by grayscale analysis. (L) Ubiquitinated CyPA measured by immunoprecipitation with anti-CyPA antibody or IgG control and immunoblotting with anti-ubiquitin antibody in H9c2 cells after overexpression of circ_005077 or not. (M) Flag-CyPA and HA-Ub were co-transfected and expressed in H9c2 cells. Ubiquitinated Flag-CyPA measured by IP with anti-Flag antibody or IgG control and immunoblotting with anti-HA antibody after overexpression of circ_005077 or not. Data were shown as mean ± SD of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, NS, no significance