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. 2002 Jun 11;99(12):7889–7894. doi: 10.1073/pnas.122246999

Figure 1.

Figure 1

Analysis of HUL-1 E3 ligase function. (A) GST (lanes 1 and 2), GST-exon 3 (lanes 3, 4, and 7), GST-680. (lane 5), GST-615 (lane 6), or no additional protein were added to the in vitro ubiquitylation reaction master mix (MM) containing recombinant E1, biotinylated Ub, and ubiquitylation buffer in the presence and absence of recombinant cdc34 and ATP and the ATP-regenerating system as indicated. Reaction mixtures were electrophoretically separated in a denaturing polyacrylamide gel and probed with streptavidin. Dots to the right of bands indicate polyubiquitylated species that are specifically present or augmented in reactions containing GST-exon 3 or GST-680. (B) Electrophoretically separated reaction mixtures containing GST (lanes 9 and 10), the indicated ICP0 chimeric protein (lanes 11–15) in addition to the master mix, or the master mix alone (lane 16) along with the indicated reaction components were probed with an Ab against GST. Reactive bands were detected with BCIP/NBT. All procedures in this and other figure legends were as described in Materials and Methods.