FIG. 7.

HnRNP U can bind TFIIH and inhibit CTD phosphorylation in vivo. (A) Coimmunoprecipitation of endogenous TFIIH complex with HA-HN proteins (lanes 1 to 8). HeLa cells were transfected with 10 μg of each expression vector, and the nuclear extract was processed as described in the text. Retention of HA-HN proteins in the TFIIH complex was monitored by immunoblotting with anti-HA. For the negative control, cells were transfected with the vector for GAL4–Oct-1, and the TFIIH complex in the GAL4–Oct-1 containing nuclear extract was isolated as described above. GAL4–Oct-1 was abundantly expressed in cells, as detected by immunoblotting with anti-GAL4 (lane 10). In the TFIIH complex, however, GAL4–Oct-1 was not present (lane 9). (B) HnRNP U inhibits CTD phosphorylation in vivo. HeLa cells (5 × 10⁶) were transiently transfected with 15 μg of expression vectors as indicated. Whole-cell lysates were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-CTD 8WG16. Ct, control (whole-cell lysate from untransfected cells).