14-3-3θ is a Binding Partner of Rat Eag1 Potassium Channels
Figure 1
Interaction of rEag1 N- and C-termini with 14-3-3θ.
(A) Schematic representation of (top) the structural topology of the rEag1 channel and (bottom) the rEag1 GST-N207 and GST-C0 fusion proteins. (B) Yeast two-hybrid assay. cDNA encoding rEag1-N207 or C0 segment was fused to the coding sequence for LexA DNA binding domain and subcloned into the pGilda vector. cDNA for the B42 transcriptional activation domain alone (Empty) or in combination with 14-3-3θ was subcloned into the pJG4-5 vector. Yeasts co-transformed with the pGilda- and the pJG4-5-based plasmids were streaked on leucine-lacking plates. (C) GST pull-down assay of in vitro translated 14-3-3θ. Pull-down products were immunoblotted with the anti-14-3-3θ antibody. Indicated to the left are the molecular weight markers (in kDa). (D,E) Cell lysates prepared from HEK293T cells expressing myc-14-3-3θ were used for GST pull-down assay with GST or the fusion protein GST-N207/GST-C0. (Left panels) Coomassie blue staining of the GST proteins. (Right panels) Immunoblotting of pull-down products with the anti-myc antibody. Input volume was 5% of that of the cell lysates for pull-down.