Fig 2: NEDD4L mediated ubiquitylation and degradation of YAP1 through binding to YAP1. (A) MDA‐MB‐231 parent cell lysates were immunoprecipitated with anti‐YAP1 antibody. The red arrow indicates the YAP1‐interacting protein identified by mass spectrometry. (B) The E3 ubiquitin ligases acting on YAP1 were predicted with the online database UbiBrowser, an integrated bioinformatics platform. The eight dots in red refer to those verified as E3 ubiquitin ligases of YAP1 in the literatures, and the 10 dots in blue are predicted as E3 ubiquitin ligases of YAP1. The capital letters in dots indicate the initial letters of E3 ubiquitin ligases‐domains: F refers to F‐box domain, R refers to RING domain, H refers to HECT domain, U refers to UBOX domain. The predicted interactions are arranged in descending order clockwise based on the confidence score. (C) NEDD4L and YAP1 were immunoprecipitated from MCF7 and SKBR3 parent cell lysates with anti‐YAP1 and anti‐NEDD4L antibodies, then detected via WB. This confirmed the interaction between YAP1 and NEDD4L. (D) Representative IHC staining images of NEDD4L and YAP1 in breast cancer tissues (n = 51) (left panel). Expression levels of NEDD4L and YAP1 were negatively correlated with one another (right panel). (E) WB analysis of NEDD4L expression in six matched pairs of primary breast cancer tissues (T) and adjacent normal tissues (N). (F) MCF7 and SKBR3 cells were transfected with NEDD4L‐siRNAs (siNEDD4L#1, siNEDD4L#2, siNEDD4L#3), and NEDD4L expression was detected with WB analysis. siNEDD4L#3 was selected for subsequent experiments. (G) Stability analysis of YAP1. MCF7 and SKBR3 cells were transfected with NEDD4L overexpression plasmid or siNEDD4L for 48 h, then exposed to CHX (100 µg/mL) for 0, 8, 10, or 12 h. WB analysis of YAP1 expression was then performed. (H‐I) YAP1 ubiquitination degradation assay. MCF7 and SKBR3 cells were transfected with NEDD4L overexpression plasmid or siNEDD4L and treated with MG132 (20 µmol/L, 6 h). YAP1 expression was detected with WB analysis. (J) Ubiquitination assays showing the effects of NEDD4L on YAP1 ubiquitination. MCF7 and SKBR3 cells overexpressing Flag‐ub were transfected with NEDD4L overexpression plasmid or siNEDD4L then treated with MG132 (20 µmol/L, 6 h). WB analysis was used to detect the ubiquitination of YAP1. Each experiment was repeated three times. Abbreviations: NEDD4L, NEDD4 like E3 ubiquitin protein ligase; IP, immunoprecipitation; IgG, immunoglobulin G; WB, Western blotting; YAP1, Yes1‐associated transcriptional regulator; IHC, immunohistochemistry; CHX, cycloheximide; Ub, ubiquitin.