Skip to main content
. 2007 Sep 17;27(22):7906–7917. doi: 10.1128/MCB.01369-07

FIG. 5.

FIG. 5.

ITSN associates with PI3K-C2β in vivo. (A) Immunocytochemical staining of endogenous ITSN (green) and endogenous PI3K-C2β (red) from LAN-1 human neuroblastoma cells. ITSN staining is shown in panels a and d. PI3K-C2β staining is shown in panels b and e. Overlap of ITSN and PI3K-C2β is shown in panels c and f. Panels g and h show LAN-1 cells stained with anti-rabbit or anti-mouse secondary antibody, respectively, as negative controls. Scale bars, 20 μm. (B) HA epitope-tagged ITSN proteins were expressed in 293T cells, immunoprecipitated with anti-HA, and then examined for association with endogenous PI3K-C2β (top panel). The bottom panel shows the expression levels for the HA-tagged ITSN proteins. Arrows indicate the positions of PI3K-C2β and the ITSN long (ITSN-L) and ITSN short (ITSN-S) isoforms. In similar experiments, antibodies to PI3K-C2α did not interact with ITSN (data not shown). (C) HA epitope-tagged ITSN truncation mutants were expressed in cells, immunoprecipitated as described for panel B, and then examined for association with endogenous PI3K-C2β. The position of PI3K-C2β is indicated by the arrow. The band above this represents a nonspecific contaminating band. The data shown are representative of the results of three independent experiments. Molecular masses (kDa) are shown on the left. (D) Mutation of the PRD of PI3K-C2β inhibits the interaction with ITSN. Full-length fragments of PI3K or PI3K NT fused to CFP were coexpressed with HA-ITSN. ITSN was immunopurified using HA antibodies (top and middle panels), and the association of PI3K was determined by Western blot analysis with green fluorescent protein antibodies (top panel). The middle panel represents a Western blot of the HA immunoprecipitates with HA antibody, demonstrating the level of HA-ITSN in each sample. The PA mutants of PI3K and PI3K NT did not interact with ITSN. The expression of each of the proteins in the cell lysates is shown in the lower panels. Molecular masses (kDa) are shown on the left. α, anti; IP, immunoprecipation; IB, immunoblot.