Fig. 6

Spatially competitive binding of ANXA1 by circNF1 decreases polyubiquitination modification of PD-L1. A Schematic representation of ANXA1 protein structure and its truncation constructs. B RIP assays validating the ectopic expression of Myc-tagged ANXA1 (full-length or short truncations) binding to circNF1 were presented using agarose gel electrophoresis (upper panel) or qRT-PCR (bottom panel) after immunoprecipitation in KYSE150 cells. C Co-IP revealing that the third repeat domain at the C-terminus of ANXA1 protein interacted with USP7 in KYSE150 cells. D Schematic representation of USP7 protein structure and its truncation constructs. E, F Co-IP showing the detailed region of USP7 bound to ANXA1 (E) or PD-L1 (F) in KYSE150 cells. G Co-IP detecting the antagonism of full-length or TRAF domain-deficient USP7 on upregulated endogenous PD-L1 ubiquitination caused by knocking down circNF1 in KYSE150 (left panel) and TE1 (right panel) cells. H Co-IP detecting the antagonism of full-length or TRAF domain-deficient USP7 on upregulated endogenous PD-L1 ubiquitination caused by ectopic expression of ANXA1 in KYSE150 (left panel) and TE1 (right panel) cells