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Figure 8


Fig. 8. SNX9 interacts via its PX domain with PtdIns(4)P-5-kinases I{alpha}, Iβ and I{gamma}, which regulate the tubulation activity of SNX9 and whose kinase activity is increased by SNX9. (A) COS-7 cell lysates were immunoprecipitated with anti-SNX9 antibody and immunoblotted (IB) with anti-SNX9 antibody or anti-PtdIns(4)P-5-kinase I{alpha} antibody. TCL, total cell lysates. (B) In vitro GST-pull down assay was performed with purified GST-fused SNX9 and its truncated mutants (2 µg each) with His-tagged PtdIns(4)P-5-kinase I{alpha} (200 ng). (C) COS-7 cells were cotransfected with GFP-tagged SNX9, SNX9-{Delta}PX or SNX9-PX with HA-tagged PtdIns(4)P-5-kinase I{alpha}, Iβ or I{gamma} and immunoprecipitation (IP) was carried out with anti-GFP antibody, followed by immunoblotting (IB) with anti-HA or anti-GFP antibody. (D) Representative images of cells that were cotransfected with GFP-tagged SNX9 and HA-tagged PtdIns(4)P-5-kinase I{alpha}, Iβ or empty vector. When HA-tagged PtdIns(4)P-5-kinase I{alpha} or Iβ were coexpressed with low levels of GFP-tagged SNX9 in COS-7 cell, tubular invaginations were significantly increased around the cell periphery. (E) The kinase dead mutant of PtdIns(4)P-5-kinase I{alpha} (D309N/R427Q) failed to increase tubulation by low levels of GFP-tagged SNX9 in COS-7 cells. (F) Coexpression of mRFP-tagged SNX9 with GFP-tagged 5-phosphatase domain of synaptojanin inhibits tubule formation even with high levels of SNX9 expression. Scale bars: 30 µm (low magnification), 2 µm (insets). (G) Recombinant PtdIns(4)P-5-kinase I{gamma}-90 (PIPK{gamma}) was pre-incubated with two concentrations of SNX9 and its activity measured using a PtdIns(4,5)P2-radiolabeling assay. At 1.0 µM SNX9, the kinase activity of PIPK{gamma} was not significantly increased, but at 5.0 µM, there was a significant 2.5-fold increase in activity (**P<0.0001). (H) PtdIns(4)P-5-kinase I{gamma} (both the 87 kDa and 90 kDa isoforms) was immunoprecipitated from rat brain cytosol and pre-incubated with two concentrations of SNX9 and its activity assayed as in G. At both 1.0 and 5.0 µM SNX9, the immunoprecipitated kinase showed a twofold (**P<0.001) and fivefold (**P<0.05) significant increase in kinase activity, respectively.