Fig. 1. Association of 14-3-3 and Kir/Gem. (A) 14-3-3 binding to Kir/Gem. (a) The Ras-like core domain (white bar), N- and C-terminal extensions (black bar) and the location of the binding sites for 14-3-3, CaM (white circles) and GTP (black circle) are shown. Mutations that affect the different binding sites are indicated. Kir/Gem-R-18 is a chimera in which the C-terminus (including S288A) was substituted by a stretch of 18 amino acids (R-18) that mediates constitutive 14-3-3 binding. (b) Cells were cotransfected with cDNAs for wt or mutated Myc-Kir/Gem proteins and GST–14-3-3 or GST–14-3-3 K49E isoform. GST–14-3-3 proteins were precipitated and associated Kir/Gem detected by western blot using a Myc antibody. (c) Cells were cotransfected with cDNAs for wt or mutated Myc-Kir/Gem proteins and GST–14-3-3 or GST–14-3-3 K49E. Kir/Gem was immunoprecipitated and associated GST–14-3-3 and endogenous 14-3-3 was detected by western blot using a 14-3-3 antibody. The IgG heavy chain, migrating just below the GST–14-3-3 in (c), is marked by an asterisk. (d and e) Cell lysates were blotted with Myc (d) or GST (e) antibodies to monitor the expression level of Myc-Kir/Gem or GST–14-3-3; st, proteins markers of known molecular mass. (B) Association of Kir/Gem with 14-3-3 isoforms. (a-c, lanes 1-7) Cells were cotransfected with cDNAs for the different GST–14-3-3 isoforms and wt or mutated Myc-Kir/Gem. GST–14-3-3 proteins were precipitated and associated Kir/Gem was detected by western blot using a Myc antibody. (a-c, lanes 8-14) Cell lysates were blotted with Myc antibodies to monitor Kir/Gem expression levels. (d) One example of cell lysates blotted with a GST antibody to monitor expression levels of the GST–14-3-3 isoforms.