Figure 1

Cby forms a stable complex with itself. (A) Coimmunoprecipitation of Myc-Cby and Flag-Cby. (B) Cby self-interaction was detected by split synthetic Renilla luciferase (hRluc) protein-fragment-assisted complementation. Cby or negative control GFP was fused in-frame to the N-terminal portion (RN) and C-terminal portion (RC) of hRluc. These expression plasmids (400 ng each) were transfected into HEK293T cells as indicated, and Renilla luciferase (Rluc) activities were measured 24 hr post-transfection. A firefly luciferase plasmid (5 ng) was co-transfected to normalize transfection efficiency. Transfections were carried out in triplicate and the means ± SD are shown.(C) Cby-Cby interactions were detectable in vivo in real time using cell-permeable ViviRen live cell substrate. Cby-RN, Cby-RC, Fos-RC and Jun-RN were transiently expressed in HEK293T cells as shown, and ViviRen was added to the tissue culture media 24 hr post-transfection for luminescence measurements. Transfections were carried out in triplicate and the means ± SD are shown.(D) Cby complex is highly stable. The immunoprecipitates were washed three times with wash buffer containing 0.135, 0.5, 1.0, 1.5 or 2.0 M NaCl, or 0.5, 1.0, 1.5 or 2.0 M urea as shown. The experiments with urea were performed in the presence of 0.135 M NaCl.