Fig. 4

RNF31 interacts with YAP and inhibits its protein stability. A Immunofluorescence imaging of RNF31 (Green), YAP (Red) and DAPI (blue) in MDAMB231 cell, scale bar 30 μm. B Western blot detecting of YAP and RNF31 protein localized in the MDAMB231 cell. Subcellular protein fractionation kit was used for cytoplasm and nucleus separation. Tubulin and Histone3 were engineered to cytoplasm and nucleus controls. C Co-IP assay revealed the association between endogenous RNF31 and YAP protein in MDAMB231 cell. D-E Schematic diagram to show wild-type and truncated RNF31 and YAP constructs used in this study. F Representative immunoblots to show the interaction between RNF31, and WT or truncated YAP as indicated assessed by immunoprecipitation (IP) with RNF31 (anti-Flag). G Representative immunoblots to show the interaction between YAP and WT or truncated RNF31 as indicated assessed by immunoprecipitation (IP) with YAP (anti-Flag). H-I Western blot detecting of YAP and RNF31 protein level in MDAMB231 and HEK293T cells in indicated treated. 10 μM MG132 was added to cells for 8 h before fractured for western-blot assays. J-M and O-P Western blot detecting of YAP protein half-life in MDAMB231 and HEK293T cell. 100 μmol/L CHX was added to cells for indicated time periods before fractured for western-blot assays. N Western blot detecting of YAP and RNF31 protein level in HEK293T cell transfected with either Flag or Flag-RNF31 or Flag-RNF31 R1/2M plasmid. The results are representative of 3 independent experiments. β-actin was engineered to the internal reference for Western blot. The data are presented as mean ± SDs. **P < 0.01 (Student’s t test)