Ubiquitination of Pol II is induced by α-amanitin in an in
vitro assay. Nuclear extracts from cells treated with
aphidicolin were incubated in an in vitro ubiquitination
reaction with pUC118-296 in the absence or presence of His-Ub and
α-amanitin (α-am). His-ubiquitinated proteins were selected on
Ni-NTA-agarose. Total (lanes 1–5), Ni-bound (lanes 6–10), and
flow-through fractions from the Ni-NTA-agarose (lanes 11–15) were
analyzed by SDS/7.5% PAGE and transferred to a poly(vinylidene
difluoride) (PVDF) membrane. The immunoblot was probed consecutively
with antibodies to the N terminus of RNA Pol II (a;
antibody N20), and the phosphorylated CTD of Pol II (b;
antibody H14). Lanes 1, 6, and 11 show untreated nuclear extracts (SM,
starting material).