Figure 3.

Mcl-1 interacts via its PEST domain with Akt at its PH domain. A, Co-IP experiments were performed in H1299 vs. H1299 Mcl-1^−/− cells using Mcl-1 or Akt antibody, followed by Western blot to measure endogenous Mcl-1/Akt interaction. IP by IgG was used as control. B and C, Co-IP experiments using Akt antibody or Mcl-1 antibody were performed in cell-free systems (recombinant His-tagged Akt and His-tagged Mcl-1 proteins in lysis buffer). D, Akt/Mcl-1 interactions were analyzed by the proximity ligation assay (PLA) in H1299 cells and H460 cells. PLA signals were detected by fluorescence microscopy as discrete spots. IgG, single Akt antibody alone or single Mcl-1 antibody alone were used as negative control. E, Schematic representation of various Flag-tagged Mcl-1 deletion mutants. F, H1299 cells were co-transfected with HA-tagged WT Akt and various Flag-tagged Mcl-1 deletion mutants, followed by co-IP using Flag antibody and Western blot using HA or Flag antibody, respectively. G, Schematic representation of various GST-tagged Akt deletion mutants. H, H1299 cells were co-transfected with Flag-tagged WT Mcl-1 and various GST-tagged Akt deletion mutants, followed by GST pull down and Western blot using Flag or GST antibody, respectively.