Pursuant to the observation that recognition of CD30 by antibodies Ki-1 and MAB229 is affected by CD30 redox state, we used flow cytometry to analyze the response of CD30 to with Trx1(CSAAA) and complexes were analyzed by anti-EGFR immunoblotting. Cellular lysate was included as control. (C) HeLa cells were transiently transfected with an expression construct for human CD30 or empty vector incubated with Trx1(CSAAA) and analyzed by immunofluorescence microscopy using CD30-and Trx1-specific antibodies (scale bar, 20 mm). (D) HeLa cells were transiently transfected with expression constructs for human CD30 or CD95, treated as described in (C) and analyzed by immunofluorescence microscopy using CD30-, Trx1-and CD95-specific antibodies (scale bar, 20 mm). (E) RMA mouse lymphoma cells were incubated with Trx1(CSAAA) or Trx1(CCAAA). Disulfide-linked Trx1 complexes were analyzed by anti-mouse CD30 (mCD30) immunoblotting under non-reducing and reducing conditions. The disulfide-linked Trx1-mCD30 complex and monomeric mCD30 are indicated. The additional band of higher molecular weight has not been further characterized but might represent a conjugate between Trx1 and a dimer of mCD30. reduction in greater detail. Addition of oxidized Trx1 did not influence antibody reactivity of CD30 (data not shown). When reduced Trx1 was applied in the absence of a regenerating system, a conformational change in CD30 could be observed, but remained incomplete (data not shown). Complete and sustained loss of antibody reactivity required a source of reducing equivalents for the oxidoreductase. Both DTT and Trx reductase (TrxR)/NADPH were found to be effective as regenerating systems. Importantly, neither DTT nor TrxR/NADPH had an effect when applied in the absence of Trx1 ( Figure 5A , middle panel and data not shown). Wild-type Grx1 ( Figure 5C , left panel) and the redox-inactive mutant of Trx1 ( Figure 5C , middle panel) did not alter the redox-sensitive CD30 epitopes. Other cell surface receptors, for example CD28, analyzed in parallel on the same cells were unaffected by Trx1 treatment ( Figure 5C, right panel) . Loss of CD30 antibody recognition typically occurred within minutes ( Figure 5D , upper panel). Testing the influence of Trx1 concentration under the same conditions, consistent effects on CD30 conformation became evident at concentrations around 100 nM ( Figure 5D , lower panel).