Materials and methods

Cell culture
BL-41, CCRF-CEM, HDLM-2, Jurkat, RMA and U937 cells were cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco). LCL-721.220 and YT cells were cultured in IMDM (Gibco), HeLa and A431 cells in DMEM (Gibco) with the same supplements.

Substrate trapping
Depending on the type of experiment, recombinant trapping mutant was applied to cytosolic preparations, human serum ultrafiltrate or intact cells. A detailed description of the different substrate trapping protocols is provided as Supplementary information.

Analysis of CD30 redox state by flow cytometry
For reduction of cell surface CD30, 2.5 × 105 cells were incubated with 5 μM Trx1 together with 200 μM DTT or 100 nM human Trx reductase (TrxR)/500 μM NADPH for 30 min at 4°C. To monitor reduction of cell surface CD30, cells were stained with anti-human CD30 monoclonal antibody MAB229 (R&D Systems), anti-human CD30 monoclonal antibody Ki-1 (Santa Cruz) or anti-human CD30 monoclonal antibody Ber-H2 (DakoCytomation) followed by incubation with R-PE-conjugated goat F(ab')2 anti-mouse Ig's (Biosource). For control, cells were stained with R-PE-conjugated anti-human CD28 monoclonal antibody (BD Pharmingen). Cells were analyzed using a FACSCalibur (Becton Dickinson) and CellQuest software.

CD30L binding assay
A total of 2.5 × 105 cells were incubated with 5 μM Trx1 (SBP-CCCCC) and 200 μM DTT for 30 min at 37°C, washed three times and incubated with 250 ng/ml recombinant CD30L-His10 (R&D Systems) for 10 min at RT. After washing, cells were stained with anti-polyHis monoclonal antibody (Sigma) followed by incubation with R-PE-conjugated goat F(ab')2 anti-mouse Ig's (Biosource).

Immunofluorescence microscopy
HeLa cells were seeded on coverslips and transfected with expression constructs using CaCl2 precipitation. After 2 days, transfected cells were fixed with 3% formaldehyde and 2% sucrose in PBS for 7 min at RT. Fixed cells were washed three times with PBS and incubated with different Trx1 constructs or recombinant CD30L (R&D Systems). Proteins were visualized using appropriate primary antibodies (Anti-CD30L polyclonal antibody (R&D Systems), anti-CD30 monoclonal antibodies Ki-1 (Santa Cruz) or Ber-H2 (DakoCytomation), anti-CD95 monoclonal antibody (a kind gift from Dr P Krammer), anti-Trx1 polyclonal antibody (M Preuss and TP Dick, unpublished) followed by FITC-conjugated anti-goat IgG, FITC-conjugated anti-rabbit IgG or TRITC-conjugated anti-mouse IgG and analyzed with a Nikon C1Si confocal microscope.