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An Interaction Network Predicted from Public Data as a Discovery Tool: Application to the Hsp90 Molecular Chaperone Machine

Figure 6

Experimental validation of the involvement of Aha1 in nucleocytoplasmic transport.

(A) PPI map of the proteins (red nodes) in the GO module “nucleocytoplasmic transport” of the Aha1-Hsp90 PPI subset of Figure 5. It contains potential and known (dashed and full line edges, respectively) Aha1 interactions. The Hsp90 client protein GR was also integrated into the predicted PPI (upper panel). The co-immunoprecipitation assays of panel B allowed the experimental confirmation and new demonstration of PPIs as indicated by red and green edges, respectively (lower panel). (B) Co-immunoprecipitation experiment demonstrating interactions between Aha1 or exportin-1 (XPO1) and components of the GO module “nucleocytoplasmic transport” shown in panel A. Flag-tagged Aha1, exportin-1, and GPR30 as an unrelated control protein were exogenously expressed in 293T cells. IPO4, importin-4; KPNA5, importin-α6. (C) and (D) Nuclear localization of GR in mouse fibroblasts with and without Aha1. Panel C shows representative micrographs of the localization of Tom.GR with our without treatment with dexamethasone (Dex) for 40 min, and an immunoblot on the right verifying the absence of Aha1 in the Aha1-null fibroblasts. Panel D shows the nuclear accumulation of Tom.GR over time in wild-type (▴) and Aha1-null (▾) cells. Nuclear localization of GR was initiated at time zero with the addition of 10 nM dexamethasone. Data points are the means with standard errors of three independent experiments where ∼100 cells were counted for each time point. *, significantly different with p<0.005.

Figure 6