(A) Purity of isolated nuclei were assessed by adhering whole cells or nuclei (prepared as described in the Materials and methods section) to coverslips using cell-tak and decorating with a combination of wheat germ agglutinin plasma membrane stain (conjugated to Alexa Fluor® 555) and DAPI nuclear stain. Scale bar=5 μm. Representative Western blot for fractionation of (B) JPR3 or (C) MW2 cell lysates. The same proportion of each cell lysate fraction (see Supplementary Figure S1 at http://www.BiochemJ.org/bj/430/bj4300215add.htm) was either Western blotted for actin or histone H1, or used for immunoprecipitation and detection of FLAG-tagged protein as described in the Materials and methods section. (D) Quantification of PtdIns5P 4-kinase IIα, actin and histone H1 levels in Western blots of MW2 cell lysate fractionations [the cytosolic fractions (C+P) were combined]. Results are means±S.E.M. (n=4 for PtdIns5P 4-kinase IIα; n=3 for actin and histone H1). C+P, cytosol; M, cytoplasmic membranes; S, sucrose cushion; N, extracted nuclei.