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. 2009 Sep 23;83(23):12068–12083. doi: 10.1128/JVI.00963-09

FIG. 6.

FIG. 6.

ANK repeats I and II of M-T5 are critical for binding Akt. The plasmids HA-Akt and a variety of myc-His-tagged M-T5 fragments (Fig. 2A) were transiently transfected into HEK 293 cells or expressed by in vitro coupled transcription-translation in the indicated (+) combinations. (A) Cell lysates were collected 48 h following plasmid transfection and assayed for binding by AlphaScreen as described in the legend for Fig. 3. (B) GST recombinant proteins were pulled down with glutathione agarose beads, and the associated proteins were resolved by SDS-PAGE and analyzed by immunoblotting with anti-myc antibody. (C) Endogenous untagged Akt protein was immunoprecipitated with anti-Akt antibody, and samples were analyzed by SDS-PAGE and probed with anti-myc antibody to detect coprecipitated proteins. The specified antibodies were used to detect the protein expression of HA-Akt, Akt-GST, endogenous Akt, and myc-His-tagged M-T5 fragments. Bands of interest are indicated with stars and arrowheads. α, anti.