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. 2012 Apr 27;7(4):e36023. doi: 10.1371/journal.pone.0036023

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A. ITSN1 was identified as a binding partner for DISC1 by Y2H [31]. However, a number of additional connections between ITSN1/2 and DISC1 were identified in our Y2H screen. Proteins shaded yellow were identified as ITSN1 or 2 binding partners by Y2H. PPFIA2 is a member of the liprinα family of scaffolds (PPFIA1-4) which bind the LAR family of transmembrane protein phosphatases and are known to form heteromeric complexes with each other [57]. Thus, ITSNs and DISC1 may interact through a heteromeric complex of PPFIA2 and PPFIA4. PDE4 (red), a known DISC1 interacting protein, was examined for interaction with ITSN1. B. Interaction of ITSN1 with phosphodiesterase 4D3 isoform. HEK293T cells grown on coverslips were transfected with VN-ITSN1-S and either VC-PDE4D3 or VC-pep as a negative control. CFP was co-transfected to mark transfected cells and only CFP-positive cells were imaged. The fluorescence pattern shown is representative of localization observed throughout the plate. Note: white scale bar represent 10? m. C. ITSN1-S and PDE4D3 interaction was confirmed by co-immunoprecipitation. HEK293T cells were transfected with VSV-tagged PDE4D3 and either VN-ITSN1-S or CFP. ITSN1 and CFP were immunoprecipitated with FLAG antibody and the specific co-immunoprecipitation of PDE4D3 was determined by Western blot analysis with αVSV antibody. Input (bottom panel labeled “Cell lysate") shows the level of PDE4D3 was expressed equally in both cell lysates. Note that an empty lane was between the CFP and VN-ITSN1-S samples on the gel. The weak signal for PDE4D3 in that lane resulted from overflow of the sample. The top 3 panels in C were from the same membrane which had been separated into the indicated size ranges and probed with the indicated antibodies. CFP and VN-ITSN1-S each possess a FLAG epitope tag. Similar results were obtained from three independent experiments. D. ITSN1 SH3 domains bind DISC1. HEK293T cells were transfected with V5-tagged full length DISC1 (DISC1 FL; aa 1–854) or a truncated DISC1 (DISC1-TR; aa 1–597) corresponding to the deletion resulting from a translocation breakpoint that disrupts the DISC1 locus [58]. GST-SH3 (encoding all 5 SH3 domains) but not GST or GST-EH (encoding both EH domains) pulls down DISC1-FL but not DISC 1-TR (top panels). Expression of DISC1 proteins in cell lysates is shown in the Western blot of cell lysates with αV5 antibody (bottom panels). Input GST fusion proteins are indicated in the Coomassie-stained gel to the far right.