Fig. 2. The Sds22p-Glc7p complex. (A) Proteins bound by protein A-tagged Sds22p (Sds22p-PrA) were recovered from extract of strain SAY1230 after affinity isolation on IgG-Sepharose followed by elution of Sds22p protein complexes with TEV protease, which cleaves between Sds22p and the protein A tag. Proteins present in the eluate when control extracts in which the protein A tag alone (PrA) was expressed are also shown. Comparison of the two lanes shows only two major bands specific to the Sds22p-PrA eluate that were identified by mass spectrometry as Glc7p and Sds22p. (B) Proteins co-purifying with protein A tagged Glc7p (PrA-Glc7p) were prepared similarly but from strain LKY150 and the two major bands indicated were identified as in A. In addition to Sds22p, several other higher molecular weight proteins ( )were present. (C) Protein extract (30 µg, lanes 1,3; or 120 µg, lanes 2,4) from a strain (LKY168) expressing HA-Glc7p and Sds22p-PrA was immunodepleted of HA-Glc7p using anti-HA antibodies and the amount of HA-Glc7p or Sds22p-PrA remaining after immunodepletion (supernatant) compared with the initial level (total) by western blot analysis. (D) Control experiment using a strain with untagged Glc7p (SAY1228), confirming that Sds22p-PrA is not depleted by the anti-HA antibodies used in (C).