Figure 3.

ARRDC1/3 negatively regulates YAP1 protein stability. A. 786-O cells were transfected with the indicated constructs. Cells were harvested for WB analyses with the indicated antibodies. B. 786-O cells were co-transfected with the indicated constructs. Cells were harvested for WB analyses. C. 786-O cells were infected with lentivirus expressing control or ARRDC1/3-specific small hairpin RNAs. After 48 h, cells were harvested for WB analyses. D. qRT-PCR measurement of the mRNA levels of ARRDC1 and YAP1 in ARRDC1-depleted cells. GAPDH mRNA was used for normalization. The mean values (S.D.) of three independent experiments are shown. *P < 0.05. E. qRT-PCR measurement of the mRNA levels of ARRDC3 and YAP1 in ARRDC3-depleted cells. F, G. 786-O cells were infected with the indicated shRNA lentivirus. After 48 h, cells were collected at various times after cycloheximide (CHX) treatment and then subjected to WB analyses. The relative intensities of YAP1 were first normalized to the intensities of actin and then to the value of the 0-h time point. H, I. 293T cells were co-transfected with the indicated constructs. Cells were harvested for WB analyses. After 24 h, cells were treated with 20 μM MG132 for 6 h. Flag-YAP1 protein was immunoprecipitated with anti-FLAG antibody. The ubiquitinated forms of YAP1 were analyzed by WB with anti-HA antibody. J, K. 786-O cells were transfected with control, ARRDC1 or ARRDC3 constructs. After 48 h, cells were harvested for qRT-PCR measurement of the mRNA levels of CYR61 and CTGF. GAPDH mRNA was used for normalization. The mean values (S.D.) of three independent experiments are shown. *P < 0.05.