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. 1999 Apr;19(4):2921–2928. doi: 10.1128/mcb.19.4.2921

FIG. 5.

FIG. 5

PKCζ, PDK-1, and p70S6K coimmunoprecipitate. (A, B, and C) 293 cells were cotransfected with HA-p70S6K (0.5 μg) and the indicated amounts of FLAG-PKCζ wt, FLAG-p38-MAPK or myc-PDK-1 or with an empty vector (pCMV5), as indicated. Cells were starved in serum-free medium for 24 h and stimulated with EGF for 30 min or not stimulated. FLAG-PKCζ wt, FLAG-p38, or myc-PDK-1 was immunoprecipitated (IP) from 33% of the total cell extract by using an anti-FLAG or anti-myc antibody, respectively. Coimmunoprecipitating HA-p70S6K was detected by immunoblotting using a C-terminal p70S6K-specific antibody. Whole-cell (W.C.) lysate was analyzed for expression of HA-p70S6K, FLAG-PKCζ wt, FLAG-p38, and myc-PDK-1. (D) 293 cells were cotransfected with FLAG-PKCζ wt (2.5 μg) and the indicated amounts of myc-PDK-1 or with an empty vector (pCMV5). myc-PDK-1 was immunoprecipitated as described above. Coimmunoprecipitating FLAG-PKCζ was detected by immunoblotting using a PKCζ-specific antibody. Whole-cell extract was also analyzed for expression of FLAG-PKCζ and myc-PDK-1. These data are representative of at least three independent transfections.