FIG 4.

Degradation of RNA Pol II by the proteasome in KSHV-reactivating cells. (A) KSHV reactivation in synchronized TREx-K-Rta BCBL-1 after treatment with a combination of TPA and doxycycline. The cell cycle was synchronized with a double thymidine block, and KSHV reactivation was induced by a combination of Dox (1 μg/ml) and TPA (20 ng/ml). The indicated molecules were stained with immune-FISH at 28 h postinduction. (B) Immunoblotting. TREx-K-Rta BCBL-1 cells were reactivated by TPA and Dox for 4 h after cell cycle synchronization. Cell lysates were prepared at different time points after reactivation, and 50-μg portions of total cell lysates were subjected to immunoblotting. The indicated proteins were probed with specific antibodies. No Dox, no KSHV reactivated cells; Dox & TPA, reactivated by TPA and Dox for 4 h. (C) Proteasome-mediated degradation of RNA Pol II. (a) TREx-K-Rta BCBL-1 cells were reactivated by TPA and Dox for 4 h after cell cycle synchronization. At 28 h after stimulation, MG132 (final concentration, 10 μM) was added to the culture media and remained another 20 h. The indicated proteins were probed with specific antibodies. (b) Bortezomib (final concentration, 16 nM) was added to the culture media after stimulation of KSHV reactivation for 4 h and treatment lasted another 44 h. At 48 h poststimulation, the cells were harvested, and cell lysates were prepared. The indicated proteins were probed with specific antibody.