FIG. 5.

Protein-lipid overlay assay showing that in vitro activation of PKCζ by ceramide leads to a loss in PIP₃ binding to PKB. (A) Nitrocellulose membranes were spotted with 1 μl of 500 pM PIP₃. Membranes were then incubated overnight at 4°C in TBST buffer containing 1 μM ATP, 4 pg of PS/ml, and 5 mM MgCl₂. This buffer also contained or lacked GST-PKB (0.5 μg/ml), GST-PKCζ (0.5 μg/ml), and/or C₂-ceramide (Cer, 100 μM) as indicated. Membranes were subsequently washed, and bound PKB was detected by probing with an anti-GST antibody. (B) To assess the importance of PKCζ activation in influencing the binding of PIP₃ to PKB, the experiment in panel A was repeated but with TBST buffer that either lacked ATP, PS, and MgCl₂ or which had been supplemented with Ro 31.8220 (Ro, 5 μM), as indicated.