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. 2002 Mar 19;99(7):4239–4244. doi: 10.1073/pnas.072068399

Figure 1.

Figure 1

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).