Figure 4

The heptad leucine residues within the coiled-coil domain are crucial for Cby homodimerization. (A) Expression levels of Cby point mutants. Lysates from HEK293T cells transfected with an equal amount (600 ng) of an expression plasmid for Flag-tagged wild-type (WT) or mutant Cby were subjected to Western blotting with an anti-Flag antibody. (B) Cell lysates were prepared from HEK293T cells transiently co-transfected with HA-CbyWT and Flag-CbyWT or individual Cby variants with all possible combinations of leucine-to-alanine mutations [single, double, triple and quadruple (4A)], and subjected to immunoprecipitation with anti-HA antibody. The immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-Flag antibody. To ensure sufficient protein expression levels, the amounts of plasmid DNA expressing Flag-tagged Cby mutants were increased by 2-fold for transfection, compared to those of Flag-CbyWT plasmid. (C) Split hRluc protein-fragment-assisted complementation assays using Cby point mutants. CbyWT or each of the indicated Cby mutants was fused in-frame to RN and RC. These expression plasmids (400 ng each) were transfected into HEK293T cells, and Rluc activities were measured as described in the legend to Figure 1B. Transfections were carried out in triplicate and the means ± SD are shown. Immunoblotting with anti-Cby antibody showed that the fusion proteins were stably expressed. Cby-RN fusion proteins appear as a doublet probably due to degradation.