Fig. 4

Presence of p27 in detergent-insoluble membrane domains. (A) Insolubility of p27 in NP-40. Western blots analysis of nonadherent peripheral blood mononuclear cells (PBMC) or PBMC activated for 24 hr in 100 nM phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA) and 1µg/ml phytohemagglutinin (PHA). Expression of proteins in total cell extracts (tot) and in membrane fractions prepared from an equivalent number of cells is shown. “NP-40” indicates the supernatent obtained after 60 min incubation in cold NP-40. The protein content of the insoluble NP-40 pellet was determined by dissolving that pellet in sodium dodecyl sulfate (SDS) buffer (laemmli). PCNA positivity indicates the activated status of the mitogen-exposed cells and that the nuclear fraction is not present in membrane preparations. Molecular weights are indicated in margins. (B) Insoluble p27 can be dissolved in octylglucoside. Western blots indicating p27 levels in the total cellular pellet after lysis in hypotonic buffer (lane 1) and in the pellet remaining after cells were incubated for 30 min in cold 1% Triton X-100 (Triton insol., lane 2). The Triton-insoluble pellet subsequently was dissolved in cold octylglucoside (OG), as described in text, and solubilized p27 (OG-supernatent, lane 3), and remaining insoluble p27 (OG pellet, lane 8) determined. Rapidly migrating variants of p27, as seen in the Triton-soluble fractions (Triton supernatents, lanes 6, 7) have been described elsewhere [Yaroslavskiy et al., submitted, and (51)].