Fig. 4

CircNF1 physically binds to ANXA1 and accelerates its degradation via the ubiquitin–proteasome pathway. A List and scoring of circNF1-binding proteins. B Secondary mass spectrometry of the top two ANXA1 scoring peptides. C Docking models of circNF1–ANXA1 complexes. D RIP assays with antibody against ANXA1 to pull down circNF1 in ESCC cells. E Confocal images of circNF1 (red) together with ANXA1 (green) in TE1 cells. DAPI staining of nuclei. Scale bar: 10 μm. F Western blotting detecting ANXA1 protein level in KYSE150 cells after knocking down or overexpressing circNF1. G Western blotting showing the effect of overexpressing circNF1 on ANXA1 protein stability in KYSE150 cells treated with 20 µg/mL CHX for the indicated times. H Western blotting analyzing ANXA1 protein level after treatment with proteasome inhibitor MG132 in circNF1-overexpressed KYSE150 cells. I, J Co-IP showing ANXA1 polyubiquitination level after circNF1 knockdown (I) and overexpression (J) in KYSE150 (left panel) and TE1 (right panel) cells