Fig. 3: YAPer-ORF interacts with GNAQ/11 to activate YAP signaling.
a–d YAPer-ORF interacts specifically with GNAQ/11. Mass spectrometry identification of ORF-interacting proteins (a). The ORF-4×HA fusion construct was transfected into HEK-293T cells for 24 h, the fusion protein was enriched with an anti-HA antibody, and the GNAQ/11 expression level was assessed via western blotting with anti-GNAQ or anti-GNA11 antibodies (b). Endogenously expressed YAPer-ORF was enriched with anti-ORF, and the GNAQ/11 expression level was assessed via western blotting (c, d). e The interaction between YAPer-ORF and the GNAQ/11-Q209L mutant was markedly elevated, as determined by the Checkmate assay. GNA11-WT and GNAQ-WT are wild-type proteins, and GNA11-Q209L, GNA11-R183C, GNAQ-Q209L, GNAQ-Q209P, and GNAQ-R183Q are mutated proteins. f YAPer-ORF overexpression significantly decreased YAP1 phosphorylation levels and activated YAP signaling activity. YAP signaling activity was determined via western blotting with anti-YAP1-p397 and anti-YAP1-p127 antibodies. RNA-FL: full-length RNA. Internal reference: GAPDH and YAP1. g YAPer-ORF overexpression upregulated the mRNA expression of YAP signaling target genes. The data in e and g are presented as means ± SDs. The error bars represent the SDs of triplicate experiments. The p values were calculated using two-tailed unpaired Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001. Western blot analysis (h, j) and quantification (i) of protein levels of YAPS127, YAPS397, YAP, LAST1, MST1, MST2, MOB1T35, MOB1 and TEAD in OMM2.3, MEL202, and MEL290 cells with overexpression/knockdown of YAPer-ORF or scramble control and treated with/without TRULI (j). k Immunofluorescence staining (left) of pYAP in heart sections from P0.5 neonatal mice and quantification (right) of the intensity of pYAP in the heart.
