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Fig. 2. Wild-type human RII{alpha}, but not RII{alpha}(T54E), co-fractionates with chromatin at mitosis. (A) Mitotic Reh-RII{alpha} cells (upper panels) and mitotic Reh-RII{alpha}(T54E) cells (lower panels) were homogenized and lysates centrifuged at 15,000 g. The sedimented material (P15) was extracted with 1% Triton X-100 and the Triton-X-100-insoluble material was treated with MNase and sedimented to produce MNase-soluble (MNase (S)) and MNase-insoluble (MNase (P)) fractions. The 15,000 g supernatants from the first centrifugation (S15) were fractionated at 200,000 g into soluble (S200) and particulate (P200) fractions. Fractions were immunoblotted using anti-AKAP95 and anti-RII{alpha} mAbs. (B) Mitotic rat PC12 cells were fractionated into P15, MNase-soluble (S) and MNase-insoluble (P) fractions, and each fraction was immunoblotted as in (A) and using anti-RIIß mAbs.