Fig. 5: USP10 and Cdh1 form a complex with ANLN in a non-competitive manner to balance ANLN levels. | Cell Death & Differentiation

Fig. 5: USP10 and Cdh1 form a complex with ANLN in a non-competitive manner to balance ANLN levels.

From: Targeting USP10 induces degradation of oncogenic ANLN in esophageal squamous cell carcinoma

Fig. 5

A–C The indicated plasmids were transfected into HEK293T cells for 48 h, then cells were harvested with EBC buffer and co-immunoprecipitated using HA or Flag magnetic beads. D The interaction between the recombinant GST-Cdh1 and His-ANLN or His-USP10 was examined using an in vitro GST pull down assay. E–G KYSE150 cells were transfected with siRNAs for 40 h, and then treated with MG132 (20 μM) for 8 h. Immunoprecipitation was used to detect the interaction between endogenous USP10, Cdh1, and ANLN. H HEK293T cells were transfected with the indicated plasmids and treated with MG132 (20 μM) for 8 h before harvesting. Ubiquitination assay was used to detect the ubiquitination level of Flag-ANLN. I KYSE150 cells were transfected with siRNAs for 48 h. The indicated antibodies were used for the western blotting. J KYSE150 cells were treated with DTB and released for different time periods. Interaction between endogenous ANLN and USP10 or Cdh1 was detected by immunoprecipitation. Cyclin B1, E2, and phospho-histone H3 (S10) were used as markers for the different cell-cycle phases. K KYSE150 cells transfected with siRNAs were synchronized and released for different time periods. ANLN levels were detected by western blotting. Cyclin B1 and phospho-histone H3 (S10) were used as markers for mitosis. L–N KYSE150 cells were transfected with siRNAs for 48 h. The indicated antibodies were used for the western blotting. All data are representative of at least three independent experiments.

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