Fig. 4
CircRNF10 interacts with ZBTB48 and prevents from degradation via inhibiting the ubiquitin- proteasomal pathway. a Venn diagram showed integrated CatRAPID, mass spectrometry and UbiBrowser results. b, c Western blotting for ZBTB48 in SYNS03 with circRNF10 knockdown followed by MKRN3 silencing (b) and circRNF10 overexpression with MKRN3 upregulation in SYNS05 (c). d RNA pulldown assays revealed the enriched MKRN3 pulled down by circRNF10 probe in SYNS03. e, f RIP assays displayed anti-MKRN3 treatment caused circRNF10 enrichment in circRNF10-silenced SYNS03 (e) and circRNF10-overexpressed SYNS05 (f). g, h Co-IP assays showed the interaction of ZBTB48 and MKRN3 in SYNS03. i-l Co-IP assays illustrated interaction efficiency of ZBTB48 and MKRN3 under the condition of circRNF10 knockdown in SYNS03 (i. j) and circRNF10 overexpression in SYNS05 (k, l). m, n Western blotting showed ZBTB48 expression in circRNF10-silenced combined with MKRN3-knockdown SYNS03 (m) or circRNF10 and MKRN3 co-overexpressed SYNS05 (l) by treated with CHX (50 μg/ml) and the half-life time (t1/2) of ZBTB48 protein was quantitative analysis. o, p. CircRNF10-silenced combined with MKRN3-knockdown SYNS03 (o) or circRNF10 and MKRN3 co-overexpressed SYNS05 (p) were treated with or without MG-132 (50 μM) for 6 h, and ZBTB48 expression was determined by western blotting. q, r In vivo ubiquitination assays showed the ZBTB48 ubiquitination levels followed by circRNF10-silenced combined with MKRN3-knockdown SYNS03 (q) or circRNF10 and MKRN3 co-overexpressed SYNS05 (r). s, t In vivo ubiquitination assays of polyubiquitin chains of ZBTB48 in SYNS03 (s) and SYNS05 (t) transfected with wild-type, K6R, K11R, K48R or K63R mutant ubiquitin plasmids. Data are presented as mean ± SD (three independent experiments). *p < 0.05; ** P < 0.01; ***P < 0.001; ns, no significance