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Fig. 4. Interaction of eIF3k with K18. (A) Yeast two-hybrid assay. Yeast strain L40 was co-transformed with LexA- and Gal4-based vectors as indicated. The transformants were tested for β-galactosidase activity (β-gal; upper panel) and for their ability to grow in medium without histidine (middle panel) as described in the Materials and Methods. Yeast grown on the –His plate and that stained positive for β-galactosidase activity indicate the interaction between proteins encoded by the two plasmids. (B) eIF3k interacts with K18 endogenously. HeLa cells were lysed with Empigen BB lysis buffer as described in the Materials and Methods, and then subjected to immunoprecipitations with antibodies to K8 and K18 (1:1 mixture of anti-K8 and -K18 antibodies; upper panel), eIF3k (lower panel) or a control IgG (both panels). The immunoprecipitates and cell lysates were resolved by SDS-PAGE and then analyzed by western blot with antibodies as indicated. The positions of immunoglobulin light chain and eIF3k are indicated in the bottom panel. (C,D) Mapping of the domains in eIF3k (C) and K18 (D) that are responsible for their interaction. Yeast strain L40 was transformed with LexA- and Gal4-based vectors as indicated and analyzed as in A. Domain organizations of eIF3k, K18 and mutants used in this study are shown in the top panel. Numbers refer to amino acid positions.
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