Figure 6.

Effect of dephosphorylation on the formation of RNP complexes under cleavage conditions. HeLa nuclear extract was dephosphorylated with 0.04 U/μg of alkaline phosphatase. Control samples were incubated with 10 mM Hepes pH 7.6 in a volume equal to that of the treated samples. Cleavage reactions were carried out for varying time intervals ranging from 0 to 30 minutes. The reactions were terminated by the addition of heparin to a final concentration of 5 mg/ml and incubated for an additional 10 minutes at 30°C. These samples were analyzed on a 4% non-denaturing poly-acrylamide gel. Lanes 2 to 4 correspond to the dephosphorylated samples incubated for 0, 5, and 30 minutes, respectively, whereas lanes 5 to 7 correspond to the untreated samples incubated for the same time intervals. The native gel showed that the formation of the pre-cleavage complex (B) and the post-cleavage complex (B′) by the dephosphorylated samples was comparable to that formed by the untreated samples (compare lane 2 with lane 5, 3 with 6, and 4 with 7).