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. Author manuscript; available in PMC: 2013 May 31.
Published in final edited form as: J Biol Chem. 2000 Jun 9;275(23):17838–17847. doi: 10.1074/jbc.M909735199

Fig. 5. Gene 33 is a cytosolic protein that interacts with 14-3-3ζ and, in a GTP-dependent manner, with Cdc42Hs.

Fig. 5

A, cytosolic localization of Gene 33. NIH3T3 cells were transfected with FLAG-Gene 33. Cells were stained with anti-FLAG and fluorescein isothiocyanate-labeled anti-mouse. Gene 33 was detected by immunofluorescence. The arrowhead indicates the nucleus. B, interaction with 14-3-3ζ. 293 cells were transfected with GST-14-3-3ζ and FLAG-Gene as indicated. GST pull-downs and anti-FLAG immunoprecipitates (IPs) were prepared and subjected to SDS-PAGE and reciprocal immunoblotting with anti-GST and anti-FLAG to detect coprecipitated proteins. C, in vivo binding of Gene 33 to Cdc42Hs. 293 cells were transfected with GST-Gene 33 and FLAG-V12-Cdc42Hs. Anti-FLAG-Cdc42Hs immunoprecipitates (IP) were prepared and subjected to washing with progressively higher concentrations of LiCl as indicated in the top panel, followed by SDS-PAGE and immunoblotting (IB) with anti-GST to detect bound Gene 33. Expression blots are shown in the bottom panel. D, the Gene 33/Cdc42Hs interaction requires the Gene 33 CRIB motif. Cells were transfected with FLAG-Cdc42Hs (V12) plus either GST-Gene 33 or HA-Gene 33 (aa 61–459). Cdc42Hs was immunoprecipitated with anti-FLAG and subjected to SDS-PAGE and immunoblotting with anti-GST or anti-HA as indicated. Crude lysates were blotted with the indicated antibodies to judge expression of the transfected proteins. E, in vitro GTP-dependent binding of Gene 33 to Cdc42Hs. Bacterially expressed GST-Cdc42Hs was loaded with either GTP-γS or GDP-βS as indicated (GTP or GDP, respectively) and incubated with extracts of 293 cells that had been transfected with Gene 33 or PAK1 (FLAG-tagged) expressed at equal levels (left) or with Gene 33 in excess (right). Crude 293 cell extracts, indicated in the figure, were subjected to SDS-PAGE and blotted with anti-FLAG to detect expression of Gene 33 or PAK1. Cdc42Hs beads were washed and subjected to SDS-PAGE and immunoblotting with anti-FLAG to detect bound Gene 33 or PAK.