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. Author manuscript; available in PMC: 2008 Aug 18.
Published in final edited form as: J Proteome Res. 2006 Jul;5(7):1636–1646. doi: 10.1021/pr0502469

Figure 4. In Vitro Phosphorylation of Recombinant Hsp70, Hsp90α and β, Grp78, Grp94, and PDI by Recombinant Active Akt.

Figure 4

1μg of recombinant human Hsp90α and β, rat Hsp70, canine Grp94, hamster Grp78, and purified PDI was incubated alone or with recombinant active Akt (400ng), in the presence of [γ-P32]ATP, at 25oC for 45 minutes. Reactions were terminated by addition of Laemmli buffer and proteins were separated by 10% SDS-PAGE (A) or 4−12% gradient gel (B). Phosphorylated proteins were visualized by autoradiography. A, autoradiograph demonstrating autophosphorylation of recombinant active Akt, alone, and minimal phosphorylation of recombinant Hsp90α and Grp94 when incubated alone in the presence of [γ-P32] ATP. Incubation of recombinant chaperone proteins with recombinant active Akt resulted in phosphorylation of Hsp90α and β, Grp94, and PDI (A) and recombinant Hsp70 (B), however, recombinant Grp78 was not phosphorylated by Akt in the in vitro kinase reaction (B).