Physical association of FLAG-tagged mutant FasL with HA-tagged wild type FasL. (a) Lanes 1, 2 and 3 contain extracts from cells transfected with the FLAG-tagged mutant FasL constructs (A320G, A530G and hgld) alone and lanes 4, 5 and 6 are from cells co-transfected with one of FLAG-tagged mutant FasL constructs and the HA-tagged wild type FasL construct. An anti-HA antibody was used for the immunoprecipitation and an anti-FLAG antibody was used for Western blot analysis. The double bands appearing at about 40 kD represent two FasL isoforms with different glycosylation patterns. (b) Western blot analysis of transfected cell lysates. The FLAG-tagged FasL with the mutations (lanes 1–3) were expressed in 293T cells, and FasL expression was detected by Western blotting with an anti-FLAG antibody.