Figure 3.

Subcellular fractionation of HeLa cells showed that nuclei contained PIPKIα and PIPKIIα. HeLa cells were disrupted and separated into nuclear and crude cytosolic (cytosol) fractions by low-speed sedimentation. The nuclei were membrane stripped with 0.8% Triton X-100, washed, and pelleted (stripped nuclei). Stripped nuclei were then treated with 0.35 M KCl and separated into soluble (nuclear extract) and insoluble (postextracted nuclei) fractions by high-speed centrifugation. Equal amounts of protein from each fraction were used to do Western blots for both PIPKIα and PIPKIIα (A). The purity of the cellular fractions was assessed by reprobing the Western blots (B) with antibodies toward proteins from the ER, cytosol, and plasma membrane (PM), as described in MATERIALS AND METHODS.