Fig. 3: circNDUFB2 physically interacts with IGF2BPs and promotes ubiquitin/proteasome-mediated degradation of IGF2BPs.
a Interaction of IGF2BP1, IGF2BP2, and IGF2BP3 with circNDUFB2. β-ACTIN was used as a negative control. b Analysis for circNDUFB2 enrichment. RIP assay was performed using the indicated antibodies in A549 cells. IGF2 was used as a positive control and TINCR was used as a negative control. n = 3 biologically independent samples. c Co-localization of circNDUFB2 (red) with IGF2BP proteins (green), respectively, in A549 cells. Scale bar = 50 μm. d RNA pull-down assays detected the interaction between circNDUFB2 and IGF2BP proteins in the indicated group. e Gene-specific m6A qRT-PCR assay detected m6A modification of circNDUFB2 in A549 cells with or without METTL3/14 knockdown. n = 3 biologically independent samples. f, g Protein levels of IGF2BPs in NSCLC cells with circNDUFB2 overexpression (f) or knockdown (g), respectively. h Protein levels of IGF2BPs in A549 cells with vector, circNDUFB2, or circNDUFB2-mutant transfection. i Protein levels of IGF2BPs in A549 cells with circNDUFB2 overexpression treated with MG132 (20 μM) for 12 h. j Immunoprecipitation detected ubiquitination modification of IGF2BPs in A549 cells. Ub ubiquitin. Data are presented as mean ± s.d. P values are calculated by unpaired two-sided t-test in b and e. Two independent experiments were carried out with similar results in b, c, d and f–j.
