Figure 6A. In vivo S-tag-PTENε pull-down analysis. Whole-cell extracts from HEK293 cells transfected with S-tag-PTENε were immunoprecipitated with S-protein beads followed by mass spectrometric peptide sequencing. S-tagged-PTEN, S-tagged-PTENα, S-tagged-PTENβ, or mock construct were introduced into HEK293 cells separately and the transfected cells were treated in the same way as described above. Cell adhesion associated protein was found in the PTENε pull-down list (left panel). Molecular function analysis of PTENε interacting proteins compared with canonical PTEN, PTENα, PTENβ, and MOCK groups B. Exogenous immunoprecipitation of PTENε with FLAG-tagged CDC42, ACTR2, VASP, and FSCN1. The plasmid expressing PTEN, PTENε, or mock carrying C-terminal S-tag and HA tag was co-transfected with FLAG-tagged CDC42, ACTR2, VASP, or FSCN1 plasmid in HEK293 cells separately. Cell lysates were incubated with S protein agarose followed by western blotting with antibodies against the FLAG tag and HA tag. G1-4 stand for immunoprecipitation group 1-4. C. Immunoprecipitation of PTENε with endogenous CDC42, ACTR2, VASP, and FSCN1. The plasmid expressing PTEN, PTENε, or mock plasmid carrying C-terminal S-tag and HA tag was transfected in HEK293 cells separately. Cell lysates were incubated with S protein agarose followed by western blotting with antibodies against CDC42, VASP, ACTR2, FSCN1, and HA tag. D. Images of immunofluorescence staining for PTENε and its interacting proteins (VASP and FSCN1). Hela PTEN-/- cells were co-transfected by a plasmid expressing C-terminal GFP-tagged PTENε with FLAG-tagged targets (FSCN1, VASP) separately, followed by staining with an anti-FLAG antibody and DAPI, and were imaged by confocal microscopy. E. The cell plasma membrane localization of PTENε is required for its interaction with downstream targets. FLAG-tagged CDC42, VASP, ACTR2, or FSCN1 plasmid was co-transfected with a plasmid expressing PTENε with or without 69-81aa deletion carrying C-terminal S-tag and HA tag in HEK293 cells. Cell lysates were incubated with S protein agarose before western blotting with antibodies against the FLAG tag and HA tag. F. PTENε protein phosphatase activity is required for its interaction with CDC42, FSCN1, VASP, and ACTR2. Wild type PTENε and PTENε with protein phosphatase activity abolished nutation (Y210L, analogous to PTEN (Y138L)) were separately co-transfected with FLAG-tagged targets (CDC42, FSCN1, ACTR2 or VASP) in HEK293 cells. Cell lysates were incubated with S protein agarose followed by western blotting with antibodies against the FLAG tag and HA tag. G. The level of phospho-VASP and phospho-ACTR2 in PTENε overexpressed Hela cells. Wild type PTENε and PTENε with protein phosphatase activity abolished mutation (Y210L, analogous to PTEN (Y138L)) carrying C-terminal S-tag and HA tag were introduced into Hela PTEN-/-cells, followed by immunoblotting with antibodies against p-ACTR2 (Thr237 and Thr238), p-VASP(Ser157), p-VASP (Ser239), p-VASP (Thr278), VASP, ACTR2, GAPDH, and HA tag. (H, I) Purified ACTR2 or VASP was phosphorylated in vitro by NIK or PKA and subsequently used in a phosphatase assay with His-PTENε or without purified PTENε protein as a control, followed by immunoblotting with antibodies against p-ACTR2 (Thr237 and Thr238) and p-VASP (Ser157). Purified proteins (PTENε, PKA, NIK, ACTR, and VASP) were detected by anti-His antibody (ZSGB-BIO, TA-02).