Fig. 1. Both Siz1 and Siz2 stimulate sumoylation of many proteins. Lysates from cells in which the indicated proteins had been tagged with the HA epitope and a His₈ tag were subjected to denaturing Ni-NTA affinity chromatography. Eluates were analyzed by SDS-PAGE and immunoblotting with antibodies against HA (lower panels) or Smt3 (top panels). SIZ genotypes are indicated over the lanes. Bands corresponding to unmodified proteins are indicated with arrowheads. Open brackets indicate sumoylated species. For Spt7, Abf1, Rsc2 and Gcn5, sumoylated species were detected with the HA Ab on a short exposure and are shown. Other substrates required long exposures of the HA blot to detect sumoylated species (not shown). A cross-reacting band is designated with an asterisk.