Fig. 5. Binding of CaM and 14-3-3 to Kir/Gem is mutually exclusive (A) and Kir/Gem in COS-1 cells is in a constitutively active, GTP-bound form (B and C). (A) Binding of 14-3-3/Kir/Gem complexes to CaM. (a) Cells were cotransfected with cDNAs for wt or mutated Myc-Kir/Gem and Flag–14-3-3. Flag–14-3-3 was first immunoprecipitated and Flag–14-3-3 (and associated Kir/Gem, if any) was eluted from the beads (under conditions that do not disrupt the 14-3-3/Kir/Gem complex; see Fig. S2a in supplementary material) and incubated with CaM beads. Flag–14-3-3 and Myc-Kir/Gem proteins associated with the CaM beads were detected by western blot by probing with both Flag and Myc antibodies. (b) The supernatant after the CaM pull-down was blotted with both Flag and Myc antibodies to detect unbound Flag–14-3-3 and Myc-Kir/Gem. (c and d) Cell lysates were blotted with Myc (c) and Flag (d) antibodies to monitor Flag–14-3-3 and Myc-Kir/Gem expression levels. (B) Ca_vß3 binds to the GTP but not GDP form of Kir/Gem. Homogenates from cells expressing Flag-Ca_vß3 were incubated with recombinant GST-Kir/Gem preloaded with either GDP-ßS (lane 2) or GTP-S (lane 3) and bound Flag-Ca_vß3 was detected by western blot using a Flag antibody. Recombinant GST served as a control (lane 1). Cell lysates were blotted with a Flag antibody to monitor Flag-Ca_vß3 protein expression levels (lane 4). (C) Binding of cellular Kir/Gem to Ca_vß3. Cells were transfected with cDNAs for wt or mutated Myc-Kir/Gem. Cell homogenates were incubated with immobilized recombinant GST-Ca_vß3 and associated Kir/Gem was detected by western blot using a Myc antibody. During the pull-down experiment, homogenates were preincubated either in the absence or in the presence of exogenous nucleotides. Recombinant GST served as a control (lane 1). Cell lysates were blotted with a Myc antibody to monitor Kir/Gem expression levels (lanes 11-13).