Figure 4.

LARP7 may occupy the catalytic center of MePCE to inhibit the latter’s capping activity. (A) Wild-type F-MePCE and the VLD-AAA mutant were affinity purified with anti-Flag beads from NEs of transfected HeLa cells under highly stringent conditions (see ‘Materials and Methods’ section) to remove any associated 7SK snRNP components. Upon their normalization to approximately the same level by anti-Flag WB (bottom panel), these two proteins were tested in 7SK capping reactions. The RNA products were isolated and analyzed by autoradiography (top panel) and EB staining (middle panel). (B) Wild-type and VLD-AAA mutant F-MePCE were affinity purified from NEs of transfected HeLa cells under relatively mild conditions to retain their associated factors, which were subsequently detected by WB and NB as indicated.