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Fig. 6. Ca_vß3 and 14-3-3 compete for binding to Kir/Gem (A) and CaM displaces Ca_vß3 from Kir/Gem (B). (A) Binding of 14-3-3, Ca_vß3 and Kir/Gem. Cells were cotransfected with cDNAs for wt or mutated Myc-Kir/Gem and GST–14-3-3, either with or without Flag-Ca_vß3. (a) GST–14-3-3 was precipitated and associated Myc-Kir/Gem and Flag-Ca_vß3 was detected by western blot by sequentially probing with Myc and Flag antibodies, respectively. (b) Flag-Ca_vß3 subunits were immunoprecipitated and associated Myc-Kir/Gem and GST–14-3-3 were detected by western blot by sequentially probing with Myc and GST antibodies, respectively. (c-e) Cell lysates were blotted with Myc (c), Flag (d) or GST (e) antibodies to monitor expression levels of Myc-Kir/Gem, Flag Ca_vß3 and GST–14-3-3, respectively. (B) CaM binding. (a) Cells were cotransfected with cDNAs for wt or mutated Myc-Kir/Gem and Flag-Ca_vß3. Flag-Ca_vß3 was immunoprecipitated. Flag-Ca_vß3 (and associated Kir/Gem, if any) was eluted from the beads (under conditions that do not disrupt the Flag-Ca_vß3/Kir/Gem complex; see Fig. S2b in supplementary material) and incubated with CaM beads. Flag-Ca_vß3 and Myc-Kir/Gem 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-Ca_vß3 and Myc-Kir/Gem. (c and d) Cell lysates were blotted with Myc (c) and Flag (d) antibodies to monitor Flag-Ca_vß3 and Myc-Kir/Gem protein expression levels.

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