FIG 4.

AJUBA LIM proteins inhibit activation of LATS by the core Hippo kinase complex and associate with LATS in proliferating cells but not growth-arrested cells in contact. (A) HEK293T cells were transfected with YAP with or without LIMD1, and the cell lysates were Western blotted with the indicated antibodies. The amount of pS127.YAP detected was controlled for the level of total YAP. The pS127YAP/total YAP ratio is shown below each lane. The amount present in cells not transfected with LIMD1 was arbitrarily set as 1. (B) HEK293T cells were transfected with different combinations of epitope-tagged plasmids expressing components of the Hippo core kinase complex, as indicated, with or without LIMD1. The cell lysates were Western blotted with the indicated antibodies. The amount of active LATS (pS872 and pT1041) in the absence of LIMD1 (equal to 1 for each set) versus the presence of LIMD1, controlled for total LATS2 protein present, was quantified. The relative amount of pS872.LATS2 or pT1041.LATS2 detected in each pair is shown below the top two panels. The amount of phospho-LATS2 species detected in cells not transfected with LIMD1 was arbitrarily set as 1 for each set. All phospho-LATS2 species amounts were normalized to total LATS2 level. (C) HEK293T cells were transfected with LIMD1 and individual components of the Hippo core kinase complex or YAP, as indicated. LIMD1 was immunoprecipitated from the cell lysates, and the bound products were Western blotted with the indicated antibodies. The left column is 10% of the amount of cell lysate used in the IP as an input control. (D) MCF10A cells grown at LD or HD were lysed, and either AJUBA or LIMD1 was immunoprecipitated. The bound products were Western blotted with the indicated antibodies. The left column shows input cell lysate controls (10% of the amount used for the IP).