Fig. 4

CircNFATC3 enhances the stability of IGF2BP3 by preventing TRIM25-mediated ubiquitination. A Western blot analysis of IGF2BP3 in SGC7901 and BGC823 cells transfected with siRNAs targeting circNFATC3. B Stability of IGF2BP3 protein in SGC7901 and BGC823 cells after circNFATC3 knockdown evaluated by cycloheximide (CHX) chase assays. C Protein expression of IGF2BP3 in SGC7901 and BGC823 cells treated with proteasome inhibitor MG132 after transfection with si-circNFATC3. D Immunoprecipitation analysis for ubiquitination modification of IGF2BP3 in SGC7901 and BGC823 cells with circNFATC3 knockdown. E–F Protein expression of IGF2BP3 in SGC7901 and BGC823 cells after TRIM25 knockdown (E) or overexpression(F). G-H Immunoprecipitation analyses of TRIM25 pulled down by IGF2BP3 (G) and IGF2BP3 pulled down by TRIM25 (HA-tagged) (H) in SGC7901 and BGC823 cells. I Immunoprecipitation analysis of TRIM25 pulled down by GFP in SGC7901 and BGC823 cells transfected with full-length of IGF2BP3 or truncations. J-K The expression of circNFATC3 (J) or TRIM25 (K) in SGC7901 and BGC823 cells transfected with si-TRIM25 and/or si-circNFATC3. L Protein levels for IGF2BP3 in SGC7901 and BGC823 cells transfected with si-TRIM25 or si-circNFATC3 alone and in combination. The Student’s t test was used to calculate p-values. ns, not significant, *P < 0.05, **P < 0.01