|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 2. 14-3-3 dimerization is required for efficient binding to Kir/Gem. (A) Coprecipitation. (a) Cells were cotransfected with cDNAs for wt Myc-Kir/Gem or the R18 chimera and GST–14-3-3 or a dimerization defective mutant (dim). GST–14-3-3 proteins were precipitated and associated Kir/Gem was detected by Western blot using a Myc antibody. (b and c) Cell lysates were blotted with Myc (b) or GST (c) antibodies to monitor Kir/Gem protein and GST–14-3-3 expression levels. (B) Coimmunoprecipitation. Myc-Kir/Gem was immunoprecipitated from cells coexpressing GST–14-3-3 or a GST–14-3-3 dimerization mutant. Overexpressed and endogenous 14-3-3 associated with Myc-Kir/Gem were revealed by western blot analysis using a 14-3-3 antibody. The asterisk indicates the band for IgG heavy chain. (C) Pull-down experiment. (a) Wt and mutated immobilized recombinant GST–14-3-3 were tested for interaction with wt or mutated Myc-Kir/Gem present in homogenates of transfected cells. Kir/Gem proteins were revealed by western blot using Myc antibody. (b) Cell lysates were blotted with a Myc antibody to monitor Myc-Kir/Gem protein expression levels.
|
