FIG. 2.
Ask10p binds Srb11p and the RNA Pol II holoenzyme in vivo. (A) Extracts were prepared from cultures under nonstress conditions (lanes 2, 4, and 6) or 30 min after oxidative stress (lanes 3 and 5) and contained Srb11p-myc, Ask10-HA (+), the Srb11pA110V mutant (A110V), or vector control (−) as indicated. Srb11p-myc immunoprecipitation (lane 6) serves as a size standard, and lane 1 controlled for nonspecific association of Srb11p-myc to protein A-Sepharose beads. (B) Extracts prepared from the ASK10-HA strain RSY869 (+) or parental control (−) were immunoprecipitated with the antibodies specific for RNA Pol II holoenzyme components Rpb1p and TFIIS as indicated. The Ask10p-specific signal and a nonspecific band (asterisk) are indicated. The negative control for nonspecific interaction of Rpb1p antibodies in a strain lacking Ask10p-HA (right panel) was overexposed to detect even minor cross-reactivity. Molecular weight markers are indicated (in kilodaltons) on the left sides of both panels.