Fig. 3

Circ_0002669 binds to MYCBP to upregulate MYCBP expression. A RNA pull-down was performed using the circ_0002669 biotin probe in U2OS cell lysates. After silver staining, MYCBP was identified as a candidate protein binding with circ_0002669 by mass spectrometry and western blot. B RNA pull-down was performed using biotin circ_0002669, and the relative expression of MYCBP was quantitated by western blotting. C RIP was performed on U2OS cell lysates using anti-MYCBP or anti-IgG, then the enrichment of circ_0002669 was detected by RT-PCR. D Circ_0002669 was co-localized with MYCBP in OS cells by FISH-IF (scale bar, 25 μm). E Stable circ_0002669-overexpressing or control OS cells were treated with MG132 (10 µM) and immunoblotted and probed with MYCBP antibody. FG Stable circ_0002669-overexpressing, -knockdown or control OS cells were treated with cycloheximide (CHX, 10 µM) and collected at different time points. Cell lysates were immunoblotted and probed with MYCBP antibody. H Ubiquitination of MYCBP in control, circ_002669-overexpressing or -knockdown OS cells were determined by immunoprecipitation with anti-MYCBP and then immunoblotting with the indicated antibodies. I Ubiquitination of MYCBP in OS cells, transfected with plasmids encoding different ubiquitin chains (K11, K48 and K63), was determined by immunoprecipitation with anti-MYCBP and then immunoblotting with the indicated antibodies. Data shown are from three independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001