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anti-CD95 monoclonal antibody (a kind gift from Dr P Krammer), anti-Trx1 polyclonal antibody (M Preuss and TP Dick, unpublished) followed by FITC-conjugated anti-goat IgG, FITC-conjugated anti-rabbit IgG or TRITC-conjugated anti-mouse IgG and analyzed with a Nikon C1Si confocal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.