Fig. 7: USP12 stabilizes YAP via inhibiting YAP K48-linked poly-ubiquitination of the YAP K315 site.
From: USP12 facilitates gastric cancer progression via stabilizing YAP
A, B Depletion of USP12 increased total ubiquitination and K48-linked poly-ubiquitination of the YAP. AGS cells were transfected with 0.5 µg HA-Ub/HA-K48/HA-K63 Ub plasmid or 0.5 µg HA-Ub/HA-K48R/HA-K63R Ub plasmid and 50 nM USP12 siRNA upon 10 µM MG132 treatment for 6 h. Cells were lysed and co-immunoprecipitated with anti-YAP antibody, and immunoblotted with the indicated antibodies. C, D USP12 Deubiquitinates YAP via K48-linked polyubiquitination. HEK-293T cells were transfected with 0.5 µg HA-Ub/HA-K48/HA-K63 Ub plasmid or 0.5 µg HA-Ub/HA-K48R/HA-K63R Ub plasmid, 1 µg Flag-USP12 and 1 µg Myc-YAP upon 10 µM MG132 treatment for 6 h. Cells were lysed and co-immunoprecipitated with anti-Myc antibody, and immunoblotted with the indicated antibodies. E, F USP12WT can be deubiquitinated YAP by K48-linked polyubiquitination, whereas USP12C48S cannot. HEK-293T cells were transfected with 0.5 µg HA-Ub/HA-K48/HA-K63 Ub plasmid or 0.5 µg HA-Ub/HA-K48R/HA-K63R Ub plasmid, 1 µg Flag-USP12WT/Flag-UAP12C48S and 1 µg Myc-YAP upon 10 µM MG132 treatment for 6 h. Cells were lysed and co-immunoprecipitated with anti-Myc antibody, and immunoblotted with the indicated antibodies. G USP12 ubiquitinates YAP through the K315 site of YAP. HEK-293T cells were transfected with 0.5 µg HA-Ub plasmid, 1 µg Flag-USP12 and 1 µg Myc-YAP WT/K76R/K90R/K97R/K102R/K181R/K204R/K252R/K254R/K280R/K315R/K321R/K342R/K494R/K497R upon 10 µM MG132 treatment for 6 h. Cells were lysed and co-immunoprecipitated with anti-Myc antibody, and immunoblotted with the indicated antibodies.
