FIG. 1.

Identification of Freud-1/Aki1 as a PDK1-interacting protein. (A) BacterioMatch II validation reporter-competent cells were transformed with a pBT vector encoding no protein (Mock) or ΔN51-PDK1 together with a pTRG vector encoding the Freud-1/Aki1 fragment (+). (B) 293T cells were transfected with the indicated plasmids encoding full-length human PDK1 and putative PDK1-interacting proteins. Immunoprecipitated (IP) proteins (top and middle) and cell lysates (bottom) were electrophoresed and immunoblotted with antibodies to FLAG or to Myc. (C) 293T cell lysates were incubated with protein A-Sepharose conjugated with normal sheep IgG (Normal), an anti-PDK1 antibody, or an anti-S6K antibody. Immunoprecipitated proteins (lanes 2 to 4) and cell lysates (Input) were electrophoresed and immunoblotted with the indicated antibodies. (D) HA-tagged recombinant (HA-rec.) Freud-1/Aki1 (lane 2) or p27^Kip1 (lane 3) protein was obtained by in vitro translation of a pHM6 vector encoding Freud-1/Aki1 or p27^Kip1. The HA-tagged protein was obtained from a pHM6 vector encoding no protein (Mock). HA-tagged proteins were immunopurified with an anti-HA agarose and then incubated with recombinant PDK1 (rec.PDK1). The reaction mixture (Input) and agarose-bound proteins (top and middle) were electrophoresed and immunoblotted with antibodies to HA or PDK1.