FIG. 3.

Biochemical evidence for cytoplasmic capping enzyme. (A) The indicated relative amounts of nuclear and cytoplasmic extracts of U2OS and Huh7 cells were analyzed by Western blotting (wb) with an affinity-purified sheep antibody against human capping enzyme (hCE). To control leakage of capping enzyme during cell lysis, the same samples were analyzed by Western blotting with antibodies to U2AF65 and histone H4 (lower panels). (B) The guanylylation activity of capping enzyme present in the indicated amounts of nuclear and cytoplasmic extracts from MEL cells, U2OS cells, and COS-1 cells was determined by incubating with [α-³²P]GTP, followed by electrophoresis on a 10% SDS-PAGE gel and PhosphorImager analysis. In the lower panel, the same extracts were analyzed by Western blotting with antibody to U2AF65. (C) Cytoplasmic extract from COS-1 cells in panel B was immunoprecipitated with nonimmune IgG or antibody to human capping enzyme. A total of 25% of the immunoprecipitates and 2.5% of input protein were analyzed by guanylylation assay.