Fig. 5.

Association of Hsp90 protein to w.t. and Ser129Ala Akt. a HA-tagged Akt (w.t. or Ser129Ala mutant) was expressed in HEK 293T cells, immobilized on resin (anti-HA Affinity Matrix, Roche), and incubated with increasing amounts of purified recombinant Hsp90. After washing, the resin-bound proteins Hsp90 and Akt were detected by Western blot (WB) with the specific antibodies. b HEK 293T cells were transfected with empty vector (−), HA-tagged w.t. Akt (w.t.), or HA-tagged Ser129Ala Akt (S129A). Lysate proteins (200 μg) were used for immunoprecipitation with anti-HA antibodies. The left panel [immunoprecipitation (IP)] shows the WB analysis of the pellets for the amount of precipitated Akt and co-precipitated Hsp90. The right panel (input) shows WB with the same antibodies of 10 μg of proteins from total lysates. c HEK 293T cells were transfected with HA-tagged w.t. Akt (w.t.) or HA-tagged Ser129Ala Akt (S129A), and treated, where indicated, with 20 μM TBB for 3 h. Then, 200 μg of lysate proteins was used for immunoprecipitation with anti-HA antibodies. Left panel (IP) shows the WB analysis of the pellets for the amount of precipitated Akt and co-precipitated Hsp90. Right panel (input) shows WB with the indicated antibodies of 10 μg of proteins from total lysates