Figure 4. PP2A co-immunoprecipitates with Akt in the presence of REDD1.

(A) Lysates from HEK293 Tet-On HA-REDD1 cells incubated with or without doxycycline (Dox) were immunoprecipitated using anti-HA-agarose followed by Western blot analysis. (B) HEK293 Tet-On HA-REDD1 cells expressing caAkt were incubated with or without Dox and phosphorylation of caAkt on Thr³⁰⁸ was assessed by Western blot analysis in three experiments (N=3); within an experiment, three independent samples were analyzed. (C) REDD1^+/+ and REDD1^−/− MEFs expressing HA-tagged caAkt were serum deprived and HA immunoprecipitates and cell lysates were subject to Western blot analysis. (D) Endogenous Akt was immunoprecipitated following treatment with thapsigargin (TG) and immunoprecipitates and lysates subjected to Western blot analysis. (E & G) HEK293 Tet-On cells were transiently transfected with empty pTRE_tight vector (EV) or pTRE_tight plasmid encoding wild-type (WT), P139A, K219A/Y222A (KYAA), or E68A REDD1 as indicated. HA-tag immunoprecipitates and lysates were subjected to Western blot analysis. Blots shown in panels A, C-E, and G are representative of results for two experiments (N=2); within an experiment, two independent samples were analyzed. (F) Alignment of REDD1 amino acid sequences with a conserved region on the PP2AB regulatory subunits required for interaction with the core enzyme.