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. 1999 May 25;96(11):6054–6059. doi: 10.1073/pnas.96.11.6054

Figure 1.

Figure 1

(A) In vitro ubiquitination of HeLa nuclear extract proteins. HeLa nuclear extract was incubated with 1 mM ATP and 1 μg of biotinylated ubiquitin (bio-Ub) as indicated. Total proteins (lanes 1–4) or avidin-bound proteins (lanes 5 and 6) were analyzed by SDS/4–20% polyacrylamide gel electrophoresis followed by Western blotting. Ubiquitinated proteins were probed either with anti-ubiquitin monoclonal antibody 1B3 (α-Ub 1B3; lanes 1 and 2) or HRP-streptavidin-biotin-complex (ABC-HRP; lanes 3–6). (B) In vitro ubiquitination of Pol II LS. HeLa nuclear extract was incubated with 1 mM ATP and 1 μg of bio-Ub as indicated. Flow-through (lanes 1–4) or avidin-bound proteins (lanes 5–8) were analyzed as described for A. Pol II LS was probed with N20, an anti-Pol II LS antibody (α-Pol II LS N20). Ubiquitinated Pol II LS (Ub-Pol II) was detected in the avidin-bound fraction as slow migrating material (lane 6).