Fig. 5

POLR2A-CUL3-RBX1 as a novel POLR2A-specific E3. A–C Reduced POLR2A ubiquitination in KO NPCs. KO and WT NPCs were cultured in the absence or presence of MG132 (10 µM). A All the ubiquitinated proteins were precipitated by anti-ubiquitin Ab or mouse IgG isotype control and then blotted with anti-POLR2A mAb 4H8 or anti-ubiquitin antibody. B,C The total POLR2A or POLR2A with high and low CTD phosphorylation was precipitated with mAb F12 (B) or mAb 4H8 (C), respectively. Their K48-linked ubiquitination was determined by blotting with anti-K48-linked ubiquitin Ab. D ARMC5 overexpression augmented K48-linked POLR2A ubiquitination. HEK293 cells were transfected with plasmids expressing FLAG-tagged ARMC5 and HA-tagged mutant ubiquitin only capable of K48-linked polyubiquitination. Proteins with K48-linked ubiquitination were precipitated with anti-HA Ab, and K48-linked POLR2A in the precipitates was detected by anti-POLR2A mAb (4H8) (upper left panel). Empty vectors were used as controls in transfection. The membrane was stripped and reblotted for total K48-linked proteins as loading control of the precipitated proteins (lower left panel). The total POLR2A and FLAG-ARMC5 expression in the lysates was detected by anti-total POLR2A mAb (F12) and anti-FLAG Ab, respectively (right panels). All experiments were repeated three times, and representative ones are shown. The bar graphs (means ± SD) in A–C summarize the results of three or more independent experiments for each panel. IP: immunoprecipitation; IB: immunoblotting. **p < 0.01; ***p < 0.001 (two-way Student’s t tests)