Figure 2. Identification of the immunogenic HSP90 sumoylation site.

Highly predicted sumoylation sites of HSP90 were single or triple mutated as indicated and tested for immunoreactivity with patients’ sera (dilution 1:10⁷) by ELISA. (A) Verification of the mutations by Western blot analysis using anti-HSP90 or anti-SUMO mAbs. (B) ELISA: As an example, immunoreactivity of one patient’s serum versus mutated and sumoylated HSP90 is shown. Patient’s serum did not react with WT HSP90 or HSP90-SUMO1 (K559R), while all other mutations did not affect the immunoreactions. (C) ELISA: all sera derived from all 27 HSP90-SUMO1–immunopositive patients showed no immunoreactivity when K559 was replaced by arginine (K559R), which lacks the respective sumoylation site. All other mutations had no effect on immunoreactivity. Each column represents an individual HSP90-SUMO1–positive patient.