The results showed that Flag-PTEN could pull down the AIF, and AIF-Flag precipitated HA-PTEN (Fig 1C and ?andD)

The results showed that Flag-PTEN could pull down the AIF, and AIF-Flag precipitated HA-PTEN (Fig 1C and ?andD).D). transition (EMT) and metastasis of malignancy cells and in orthotopically implanted xenografts. Accordingly, the expression of AIF is usually correlated with the survival of human patients with cancers of multiple origins. These results identify Mouse monoclonal to CRTC2 PTEN as the substrate of AIF oxidoreductase and reveal a novel function for AIF in controlling tumor metastasis. and in orthotopically implanted xenografts. Results Direct conversation of AIF with PTEN protein To explore the potential AIF-interacting proteins, human embryonic kidney 293T cells were transfected with the vacant or Flag-tagged AIF-expressing plasmids, and cell lysates were immunoprecipitated (IP) by anti-Flag antibody. The precipitates were separated on SDSCPAGE, followed by in-gel digestion and LCCMS/MS analysis (Fig 1A). Totally, 105 AIF-interacting candidates were identified (data not shown), which included four known AIF-interacting proteins: X-linked inhibitor of apoptosis (XIAP) 13, E3 ubiquitinCprotein ligase CHIP 14, optic atrophy 1 (OPA1) 15, and mitochondrial import factor CHCHD4 16. The interactions of AIF with XIAP and OPA1 were confirmed by co-IP-based immunoblots (Fig 1B), supporting the specificity and effectiveness of our co-IP assay. Of great interest, PTEN protein was among these AIF-interacting proteins, which could also be confirmed by immunoblotting with anti-PTEN antibody (Fig 1B). To consolidate the AIFCPTEN conversation, AIF and/or Flag-PTEN, or hemagglutinin (HA)-PTEN and/or AIF-Flag were exogenously expressed in 293T cells followed by IP with anti-Flag antibody. The results showed that Flag-PTEN could pull down the AIF, and AIF-Flag precipitated HA-PTEN (Fig 1C and ?andD).D). The conversation between endogenous AIF and PTEN was also found in colon cancer cell collection SW620 cells but not in PTEN-deficient prostate malignancy cell collection LNCaP cells (Fig 1E). Furthermore, glutathione S-transferase (GST) pull-down assay showed that this recombinant GST-tagged AIF, but not GST alone significantly pulled down His-tagged PTEN (Fig 1F), supporting a direct conversation of AIF with PTEN. Open in a separate window Physique 1 AIF and its isoforms interact with PTEN A Workflow for identification of AIF-interacting proteins. BCD 293T cells were transfected with AIF-Flag and HA-tagged XIAP (B), AIF and Flag-tagged PTEN (C), or Flag-tagged AIF and HA-tagged PTEN (D). Co-IP was performed with M2 beads followed by Western blots for the indicated proteins. Note: Input blot in (C) was initially detected with a rabbit anti-AIF antibody followed by HRP-conjugated anti-rabbit IgG. Then, the blot without stripping was used to detect Flag-PTEN with a mouse anti-Flag antibody followed by HRP-conjugated anti-mouse IgG. E Cell lysates from LNCaP and SW620 cells were immunoprecipitated with anti-PTEN antibody, and precipitates/input were detected by Western blots. F (1S,2S,3R)-DT-061 Bacterially expressed GST or GST-AIF protein was incubated with His-PTEN, followed by GST pull-down and Western blots for His and GST. The vacant arrowhead points (1S,2S,3R)-DT-061 to a non-specific band. GCI Schematic illustrations of PTEN fragments (G). Flag-PTEN-N or Flag-PTEN-C were transfected into 293T cells together with AIF, followed by co-IP with M2 beads (H) or anti-AIF antibody/IgG (I). The precipitates were detected by Western blots. The vacant arrowhead points to a non-specific band. PPase, phosphatase. J Schematic illustrations of AIF fragments, isoforms, and deleted mutants. MLS, mitochondrial localization transmission; IMSS, intermembrane space-targeting transmission. K GST alone or GST fusion proteins were incubated with extracts prepared from 293T cells transfected with Flag-PTEN-N, and GST pull-downs were analyzed by Western blots with antibodies against Flag and GST. Arrows point to the indicated GST or GST fusion proteins. L SW620 cells were separated (1S,2S,3R)-DT-061 into cytosol (Cyto) and mitochondria (Mito) fractions, followed by Western blots. The vacant arrowhead indicates an unknown band. Domain name mapping of AIFCPTEN conversation To map the domains of PTEN involved in its conversation with AIF, the Flag-tagged N-terminal fragment with phosphatase activity (PTEN-N) and C-terminal fragment (PTEN-C) of PTEN (Fig 1G) were transfected into 293T cells together with AIF, followed by co-IP with anti-Flag antibody. As depicted in Fig 1H and ?andI,I, PTEN-N but not PTEN-C pulled AIF down, and anti-AIF antibody pulled PTEN-N down but not PTEN-C, proposing the N-terminal phosphatase domain name of PTEN is required for its conversation with AIF protein. To define the PTEN-binding domain name of AIF, the recombinant GST-tagged full-length AIF (AIF-FL) and its three fragments, including.