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Nuclear localization of phosphatidylinositol 4-kinase ß

Petra de Graaf, Elsa E. Klapisz, Thomas K. F. Schulz, Alfons F. M. Cremers, Arie J. Verkleij and Paul M. P. van Bergen en Henegouwen*

Molecular Cell Biology, Institute of Biomembranes, Universiteit Utrecht, The Netherlands



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  		Fig. 1. Lipid kinase activity in total cell lysate and detergent insoluble fraction. (A) Total cell lysate (10 µg) and detergent insoluble fraction (DIF) (50 µg) from NIH 3T3 cells were applied to an in vitro lipid kinase assay using PtdIns as substrate. After an incubation of 20 minutes at 37°C, the lipids were extracted, separated on TLC and the PtdIns(4)P spots were quantified (total cell lysates, s.e.m., n=3; DIF, s.e.m., n=4) (*P<0.01). Adenosine, 200 µM; wortmannin, 1 µM; LY294002, 10 µM. (B) Total cell lysates (10 µg) from NIH 3T3 cells were incubated with increasing amounts of adenosine and applied to an in vitro kinase assay. Results are expressed as the percentage of control activity without adenosine.

 


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  		Fig. 2. Western blot analysis of subfractions of the detergent insoluble fraction (DIF) of NIH 3T3 cells. Proteins from cell equivalents of cell lysate (CL), total DIF (DIF), nuclear matrix (NM) and actin-enriched fraction (AF) were separated on 10% SDS-PAGE and subsequently western blotted and incubated with antibodies directed against caveolin-1 (A), GM130 (B), actin (C) and lamin A/C (D).

 


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  		Fig. 3. Lipid kinase activity in actin filaments and lamina-pore complexes. Proteins from actin filaments and lamina-pore complexes (both 50 µg) from NIH 3T3 cells were used in a lipid kinase assay as described in the legend to Fig. 1, and the PtdIns(4)P spots were quantified (actin filaments: s.e.m., n=8; lamina-pore complexes: s.e.m., n=7) (*P<0.01). Adenosine, 200 µM; wortmannin, 1 µM; LY294002, 10 µM.

 


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  		Fig. 4. PI4K230 is absent in NIH 3T3 and COS-1 cells. Cells were either mock transfected or transfected with cDNA encoding myctagged PI4K230. Proteins from cell lysates from equal amounts of cells were separated on 6% SDS-PAGE for PI4K230 and on 8% SDS-PAGE for PI4Kß and subsequently blotted and incubated with antibodies directed against myc epitope (9E10), PI4K230 and PI4Kß. Anti-PI4K230 was prepared as described in the Materials and Methods. Epitope-tagged PI4K230 was used as a control to demonstrate the expression of PI4K230 in COS-1 cells.

 


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  		Fig. 5. The presence of PI4Kß in the nuclear fraction. Proteins from cell equivalents of cell lysate (CL), post nuclear supernatant (PNS) and five times cell equivalents of membrane-depleted nuclei (N) were separated on 8% SDS-PAGE and subsequently western blotted and incubated with antibodies directed against tubulin, PI4Kß and the myc-epitope (9E10). 3T3, proteins from mock-transfected NIH 3T3 cells; CHO, proteins from mock-transfected CHO cells, and CHO + mPI4Kß, CHO cells transfected with epitope-tagged PI4Kß (myc).

 


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  		Fig. 6. PI4Kß accumulates in the nucleus upon leptomycin B treatment. COS-1 cells were mock transfected (A,B) or transfected with myc-PI4Kß (C,D). Cells were left untreated (control, A,C) or treated for 16 hours with 10 ng/ml leptomycin B (+LB, B,D). After incubation, cells were fixed and stained with anti-PI4Kß followed by GAR-Cy3 (A,B) or with an anti-myc antibody (9E10) followed by GAM-Cy3 (C,D).

 

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