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. 2022 Dec 5;13(12):1021. doi: 10.1038/s41419-022-05474-5

Fig. 5. USP36 expression is positively correlated with YAP expression and interacts with YAP in ESCC cells.

Fig. 5

A Immunostaining demonstrated the colocalization of USP36 (green) and YAP (red) in ECA109 cells. Blue denoted cell nuclei. Scale bar, 10 µm. B Representative immunoblots to show the interaction between USP36 and YAP as assessed by immunoprecipitation (IP) for USP36 or YAP, compared to isotype control (IgG). C Control or USP36-slienced ECA109 cells were treated with MG132 (10 μM) for 24 h. YAP and USP36 protein were measured by immunoblotting. Depletion of USP36 decreased YAP protein levels, and this effect could be attenuated by MG132. D USP36 depletion decreased the YAP protein half-life in ECA109 cells. The cells were treated with 100 µmol/L CHX for the indicated time before being collected for western blot assays. Quantitative analysis of halftime of YAP and TAZ protein in (D). E Representative immunoblots to show the interaction between USP36 and WT or truncated YAP as indicated assessed by immunoprecipitation (IP) with USP36 (Flag). YAP binded to USP36 at its WW domain. (Top panel) Schematic diagram of vectors expressing Myc-tagged wild-type or serial deletion mutants of YAP. (Bottom panel) The WW domain of YAP interacted with USP36. F Representative immunoblots to show the interaction between YAP and WT or truncated USP36 as indicated assessed by immunoprecipitation (IP) with YAP (anti-Myc). USP36 binded to YAP at its UPS domain. (Top panel) Schematic diagram of vectors expressing Flag-tagged wild-type or serial deletion mutants of USP. (Bottom panel) The USP domain of USP36 interacted with YAP. G, H ECA109 cells were transfected with vector, WT, or truncated USP36 and then subjected to immunoblotting to examine YAP protein level or qPCR to examine YAP target gene expression. The USP domain was essential for USP36 to maintain YAP protein levels and YAP target gene expression. I, J ECA109 cells were transfected with vector, WT, or USP36 mutant(C131A) and then subjected to immunoblotting to examine YAP protein level or qPCR to examine YAP target gene expression. The C131A mutation of USP36 impaired its ubiquitination activity and reduced the ability of USP36 to maintain YAP protein levels and YAP target gene expression in ECA109 cells. **P < 0.01, ***P < 0.001.