Figure 3.

K8 G62C mutation enhanced K8/K18 binding with Akt. (A) BHK-21 cells were transfected with Akt, K18 WT, and one of the indicated K8 mutants identified in patients with liver diseases. The cell lysates were immunoprecipitated against K8/K18, and the K8/K18 immunoprecipitates were immunoblotted with antibody against Akt to verify the K8/K18–Akt interaction. The graph represents the means ± S.E. from three independent experiments. * indicates p < 0.05. (B) BHK-21 cells were transfected with Akt, K8 WT, and one of the indicated K18 mutants found in patients with liver diseases or the caspase-resistant mutant. The K18 D238/396E (DEDE) mutant is resistant to caspase-induced K18 cleavage. The K8/K18 immunoprecipitates were prepared and immunoblotted as described in panel A. The graph represents the means ± S.E. of three independent experiments. (C) BHK-21 cells were transfected with Akt WT, K18 WT, and one of K8 constructs (K8 WT, K8 G62C, or K8 S74 phospho-deficient mutant (K8 S74A). The cells were treated with OA, and then K8/K18 immunoprecipitates and total lysates were immunoblotted with antibody to the indicated epitopes. (D) BHK-21 cells or transfected BHK-21 cells with K8/K18 WT were treated with cycloheximide (CHX). The cells were harvested after 12, 24, 36, 48, and 60 h, and the cell lysates were immunoblotted with antibody against Akt or actin. The graph represents the means ± S.E. of three independent experiments. (E) BHK-21 cells were transfected with Akt WT, K18 WT, and one of K8 WT or K8 G62C. The transfected cells were harvested at 12 h-intervals after CHX treatment. The data are represented as the means ± S.E. from three independent experiments. (F) The transfected BHK-21 cells were prepared as described in panel C. Akt activity was examined by immunoblotting of cell lysates with antibody against phosphorylated Akt T308. The relative Akt activity is shown in the graph as the means ± S.E. from three independent experiments. ‘n.s.’ indicates ‘not significant’.