Fig 1: USP10 interacts with ANLN and positively regulates ANLN protein level.A Flow chart of an in vivo screen to identify candidates that interact with ANLN in KYSE150 cells. B Potential ANLN-interacting DUBs were identified by mass spectrometry analysis. C The indicated plasmids were transfected into HEK293T cells for 48 h, and then harvested with EBC buffer for co-immunoprecipitation. D-E Interaction of endogenous ANLN and USP10 in KYSE150 and KYSE30 cells was detected by co-immunoprecipitation. F, G Schematic illustration of USP10 structure (F). The indicated plasmids were transfected into HEK293T cells for 48 h, then cells were lysed and subjected to immunoprecipitation with HA magnetic beads (Thermo Fisher Scientific, 88837) (G). H, I Schematic illustration of ANLN structure (H). The indicated plasmids were transfected into HEK293T cells for 48 h, then cells were lysed and subjected to immunoprecipitation with HA magnetic beads (I). J The interaction between recombinant GST-USP10 and His-ANLN mutants was examined using an in vitro GST pull down assay. K, L ESCC cells were treated with different concentrations of spautin-1 (MedChemExpress, HY-12990) or HBX19818 (MedChemExpress, HY-17540) for 24 h, and then western blotting was used to detect the protein levels of ANLN and USP10. M KYSE150 and KYSE510 cells were transfected with siRNAs for 48 h. The indicated antibodies were used for the western blotting. N KYSE150 cells stably expressing HA-vector, HA-USP10 or HA-USP10-CA were transfected with USP10 3' UTR siRNA, and the indicated antibodies were used for the western blotting. All data are representative of at least three independent experiments.