FIG 6.

In proliferating cells, AJUBA LIM proteins sequester LATS in a Hippo core kinase complex that does not contain YAP. (A) HEK293T cells were transfected with epitope-tagged Hippo core kinase complex plasmids, with or without LIMD1, as indicated. The cells were lysed, MST2 was immunoprecipitated, and the bound products were Western blotted with the indicated antibodies. The left column is the input controls (10% of the amount of cell lysate used for IP). (B and C) Quantification of the relative amounts of LATS2 (B) and MOB1A (C) in MST2 IP in the presence of increasing amounts of LIMD1. The value in cells not transfected with LIMD1 was arbitrarily set as 1. (D) The same experiment as in panel A, but all the cells were also transfected with a YAP-expressing plasmid. (E) HEK293T cells were transfected with epitope-tagged Hippo core kinase complex plasmids with or without LIMD1, as indicated. The cells were lysed, LATS2 was immunoprecipitated, and the bound products were Western blotted with the indicated antibodies. The left column is the input controls (10% of the amount of cell lysate used for IP). (F) Quantification of the relative amounts of YAP in LATS2 IP in the absence (CTL) or presence of LIMD1. The value in cells not transfected with LIMD1 (CTL) was arbitrarily set as 1. (G) All HEK293T cells were transfected with epitope-tagged LATS and then MOB1A, LIMD1, or YAP individually. LATS2 was immunoprecipitated from the cell extracts, and the bound products were Western blotted with the indicated antibodies. **, P < 0.01; *, P < 0.05. All the quantified experiments were performed at least 3 times, and a representative example is shown. The data are presented as means and SD.