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. Author manuscript; available in PMC: 2009 Sep 9.
Published in final edited form as: Biochem J. 2008 Jan 15;409(2):525–533. doi: 10.1042/BJ20071263

Fig. 5. Zizimin1 PH domain mediates membrane targeting.

Fig. 5

A NIH 3T3 cells were transfected with HA-tagged full-length (FL) zizimin1 or a mutant lacking the PH domain (zizimin1Δ287). Cells were lysed in hypotonic buffer and separated into cytoplasmic (c) or particulate (p) fractions, which were analyzed by Western blotting with anti-HA. The particulate fractions represent 5x more cell equivalents than the cytoplasmic fractions.

B GFP-tagged zizimin1 PH domain (zizimin1172–282) or K183A mutated PH domain were expressed in NIH 3T3 cells and their distribution between the cytoplasmic and particulate fractions analyzed as in A. The blot was probed with anti-GFP Ab. The particulate fractions analyzed represent 10x more cell equivalents than the cytoplasmic fractions.

C Lipid dot blots (100 pmol/spot) were probed with 0.5 µg/ml Flag-tagged zizimin1 PH domain (35 nM) or the full-length protein (2.1 nM). Proteins were detected using anti Flag Ab. Control blots probed with unrelated flag-tagged fragments displayed no signal (not shown). LPA – lysophosphatidic acid, LPC – lysophosphatidylcholine, PI – phosphatidylinositol, PI3P – phosphatidylinositol-3-phosphate, PI4P - phosphatidylinositol-4-phosphate, PI5P - phosphatidylinositol-5-phosphate, PE – phosphatidylethanolamine, PC – phosphatidylcholine, S1P – sphingosine-1-phosphate, PI(3,4)P2 - phosphatidylinositol-3,4-biphosphate, PI(3,5)P2 - phosphatidylinositol-3,4-biphosphate, PI(4,5)P2 - phosphatidylinositol-4,5-biphosphate, PI(3,4,5)P3 - phosphatidylinositol-3,4,5-triphosphate. PA - phosphatidic acid, PC – phosphatidylcholine.