Fig. 3
CircRNF10 interacts with ZBTB48 and prevents from degradation via inhibiting the ubiquitin- proteasomal pathway. a Venn chart of circRNF10 downstream target ZBTB48 screening. b Silver staining for circRNF10 interacting proteins in SYNS03 separated by SDS-PAGE. c Co-localization of circRNF10 (RED) and ZBTB48 (GREEN) respectively, in SYNS03 and SYNS05. Scale bar = 100 μm. d RNA pull-down assays in SYNS03 displayed the circRNF10 probe pulled down ZBTB48 proteins. e RIP assays showed anti-ZBTB48 treatment leaded to circRNF10 enrichment in circRNF10-silenced SYNS03. f RNA–protein interaction between circRNF10 and ZBTB48 predicted by the CatRAPID algorithm (top) and the diagrams of Flag-tagged ZBTB48 truncations (bottom). g RIP assays were performed in SYNS03 transfected with the truncated mutant vectors to validate the binding domain of ZBTB48. h Left, western blot showed the expression of Flag-tagged ZBTB48 truncations of SYNS03 transfected with the indicated vectors; Right, RNA pulldown assays revealed the enriched ZBTB48 truncations pulled down by circRNF10 probe in SYNS03. i qPCR assays of the mRNA expression of ZBTB48 in circRNF10-overexpressed SYNS05 and SYNS06. j Western blotting showed the protein level change of ZBTB48 after circRNF10 overexpression in SYNS05 and SYNS06. k, l The circRNF10-silenced SYNS03 (k) or overexpressed SYNS05 (l) were treated with CHX (50 μg/ml) and the expression of ZBTB48 protein was detected by western blotting and the half-life time (t1/2) was quantitative analysis. m, n The circRNF10-silenced SYNS03 (m) or overexpressed SYNS05 (n) were treated with or without MG-132 (50 μM) for 6 h, and ZBTB48 expression was detected by western blotting. o, p Ubiquitination assays showed the ZBTB48 ubiquitination levels in GSCs followed by circRNF10 silencing (o) or overexpressed (p). The GSCs were pretreated with MG132 (50 μM) for 6 h. All data are expressed as the mean ± SD (three independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001; ns, no significance