The method used to isolate nuclei has a direct effect on the subnuclear association of the v-myb product, p48v-myb and nuclear actin. Analysis of nuclei subjected to various isolation procedures showed that disruption of native nuclear structure during hypotonic treatment resulted in dissociation of p48v-.Yb from the nuclear matrix.The product of the oncogene v-myb of avian myeloblastosis virus (AMV) in AMV-transformed myeloblasts is a 48,000-Mr nuclear protein, p48V--myb (4,13). To help define the molecular role of p48v-myb in leukemogenesis, several investigators have sought to identify and define its nuclear interactions (1,2,8,12,14,15). The association between p48v-myb and nuclear elements has been analyzed in fractionated nuclei isolated from AMV-transformed cells (BM2) by two different strategies (1,8). One isolation procedure has been previously described (1, 3), in which nuclei are obtained from cells lysed under isotonic conditions in buffer containing 5 mM NaH2PO4 (pH 7.4), 50 mM NaCl, 150 mM sucrose, 5 mM KCl, 2 mM dithiothreitol (DTT), 1 mM MgCl2, 0.5 mM CaCl2, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1% Kyro EOB detergent (isotonic nuclei). After extraction of these isotonic nuclei with isotonic Nonidet P-40 (NP-40) buffer (0.5% NP-40, 10 mM NaH2PO4, pH 7.4, 120 mM NaCl, and 0.1 mM PMSF) (1), brief digestion with micrococcal nuclease at 37°C, and then extraction is 2.0 M NaCl (high salt), p48v-myb was removed from the nucleus in varying amounts in three distinct fractions: approximately 29% was contained in nucleoplasm, 7% was in nuclease-sensitive chromatin, and 64% was associated with the nuclear matrix/lamina complex (1). The other procedure we used to isolate nuclei relies on osmotic and shear force to separate the plasma membrane by disrupting cells which had been preincubated in hypotonic buffer (RSB: 10 mM NaCl, 10 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], pH 7.4, 1.5 mM MgCl2, 0.1% sodium deoxycholate [DOC], and 0.1 mM PMSF) with a Dounce homogenizer as described by Evan and Hancock (8).After fractionation of these nuclei (hypotonic nuclei) by digestion with DNase I at low temperature (4°C) followed by extraction in high salt, most or all of p48v-myb was eluted from nuclei along with soluble nucleoplasm and chromatin (Fig. 1A) as previously described (8). However, when these hypotonic nuclei were transiently exposed to temperatures near 37°C during nuclease digestion, most or all of p48v-myb remained associated with the resulting insoluble nuclear complex (Fig. 1A) 92138-9216. isolated by this hypotonic procedure is an important factor in nuclear subfractionation. These results could suggest that localization of p48V-myb within the nuclear matrix fraction is an artifact induced by a temperature-dependent insolubilization of nuclear proteins.Alternatively, the isolation procedure alone, not the subsequent method of subnuclear fractionation, may affect the associations of some nuclear proteins, including p48vmYb. In nuclei isolated under hypotonic conditio...