Figure 4.

Nuclear and cytosolic PIPKs exhibited similar kinase activities toward PI4P and PI3P as substrates. PIPKIα (A) and PIPKIIα (B) could be selectively immunoprecipitated from HeLa cytosol or nuclear extract. (A) PIPKIα was immunoprecipitated from either 200 μg of cytosol or 100 μg of nuclear extract with the rabbit anti-PIPKIα polyclonal antibody, followed by Western blotting with the PIPKIα C-terminal isoform-specific antibody. (B) The goat N19 peptide PIPKIIα isoform-specific antibody was used to immunoprecipitate PIPKIIα from 130 μg of cytosol or 65 μg of nuclear extract. PIPKIIα was then detected by blotting with the rabbit anti-PIP5KIIα polyclonal antibody. (C) PIPKIα and PIPKIIα, immunoprecipitated from 400 μg of cytosol or 200 μg of nuclear extract, were assayed for lipid kinase activity toward either PI4P or PI3P. The assays shown are representative of two or three immunoprecipitations from two different nuclear preparations. As controls, mock immunoprecipitations (mock IP) were performed in the absence of the antibody, and a sample of the antibody used for the immunoprecipitation was Western blotted (IgG).