Figure 5
From: A novel mechanism of cell growth regulation by Cell Cycle and Apoptosis Regulatory Protein (CARP)-1
CARP-1 threonine 667 is a target of H89 signaling, regulates CARP-1-TAZ binding, and CARP-1-dependent apoptosis. (A) Cells transfected with plasmid encoding myc-His-tagged CARP-1 (651-759) mutant were either untreated (Control) or treated with H89 for indicated dose and time, and cell lysates were prepared, and protein lysate subjected to immunoprecipitation using phospho-theronine antibodies (noted as P-Thr Ab), and protein lysate (100 μg, lane indicated with -) along with immunoprecipitates (lanes indicated with +) analyzed by western blotting with anti-myc-tag antibody. (B) Cells were transfected with plasmid encoding flag-tagged TAZ (1-120) mutant [indicated as pEBG-TAZ (1-120)] in combination with plasmids expressing noted myc-His-tagged CARP-1 mutant proteins. The cell lysates in lanes 2, 3, and 4 were subjected to immunoprecipitation using anti-Gst tag antibodies essentially as in figure 4B. Protein lysate and immunoprecipitates analyzed by western blotting as in panel A. Presence of CARP-1 (651-759) proteins is indicated on the left side of each panel. (C) Cells were either untransduced (Control), or transduced with retroviruses expressing vector or myc-His-tagged CARP-1 mutants. (D) Cells were transduced with retroviruses encoding vector plasmid or wild-type TAZ followed by treatments with H89 for noted dose and time. Protein lysates in both panels C and D were assayed for apoptosis essentially as in figure 1D. Columns in histograms represent data from 2 independent experiments; bars, SE. Data in panels C and D were analyzed using a two-tailed Student t test. For panel C, *P < 0.005, while for panel D, *P < 0.0087, compared to the corresponding vector controls.