Fig. 3

CircECE1 interacts with c-Myc to prevent SPOP-mediated degradation. a. Identification of the regions of the c-Myc protein that interact with CircECE1. The fragments of the c-Myc protein are illustrated (left); the interaction of c-Myc protein regions with CircECE1 in 293 T cells was confirmed using a RIP assay (right). The interaction of CircECE1 with c-Myc and SPOP was verified using a RIP assay. b-c. Schematic diagram of CircECE1 WT and MUT (left); RNA immunoprecipitation experiments were performed using anti-c-Myc antibodies in 143B cells by qRT-PCR (b) and RT-PCR(c), and specific primers were used to detect CircECE1 or GAPDH; n = 3. d-e. The effect of CircECE1 WT/MUT on the expression of c-Myc protein levels in OS cells as assessed by WB (d) and IF (e). f. The effect of CircECE1 WT/MUT on the expression of c-Myc mRNA levels in OS cells as assessed by qRT-PCR. g. The effect of CHX treatment on the change in the c-Myc protein level mediated by CircECE1 WT and MUT overexpression as detected by western blotting. h. The effect of Bortezomib treatment on the change in the c-Myc protein level mediated by CircECE1 knockdown as detected by western blotting. i. The ubiquitination levels of c-Myc in CircECE1 WT or MUT cells as measured by IP experiments. Bortezomib (250 nM) and NEM (5 μm)(Protease inhibitor) or PR-619 (100 nm)(Non-selective deubiquitinating enzyme inhibitor) were added for 6 h. j. The ubiquitination levels of c-Myc in CircECE1 knockdown cells as measured by IP experiments. Bortezomib (250 nM) and NEM (5 μm) or PR-619 (100 nm) were added for 6 h. k. The effect of SPOP knockdown on the change in the c-Myc protein level mediated by CircECE1 knockdown as detected by western blotting. l. Sequence alignment of c-Myc with the SPOP binding motif (SBC) in known SPOP substrates. m. The interaction of CircECE1 WT and MUT with c-Myc in OS cells was verified using a RIP assay. n. The interaction of c-Myc with SPOP was verified using a Co-IP assay. o. Role of SBC1 and SBC2 in the direct interaction of SPOP with c-Myc. 293 T cells were transfected with expression vectors for SPOP WT and FLAG- c-Myc or with c-Myc that was mutated within the SBC (ΔSBC) 1 or 2 for 24 h. Bortezomib (250 nM) was then added for 6 h. Total cell lysates were prepared, and IP experiment was conducted using anti-FLAG or anti-SPOP antibodies. p. 293 T cells were transfected as indicated with HA-SPOP WT and Flag-c-Myc or Flag-c-Myc ΔSBC1 for 24 h. Immunoblot analyses were conducted to detect for Flag-tagged c-Myc, HA-tagged SPOP, and β-actin