FIGURE 1.

TfR2 interacts with CD81. HEK293 cells were transiently transfected with TfR2 constructs and either CD81-Myc or empty vector (EV). Cells were lysed in IP lysis buffer 48 h post-transfection, 10% of the lysate was reserved (input), and the remaining 90% was subjected to IP with rabbit anti-Myc coupled to beads or anti-TfR2 antibody and protein A-agarose beads. Co-immunoprecipitated proteins were detected with mouse anti-TfR2 and anti-Myc antibodies. A, full-length TfR2 interacts with CD81. TfR2 only co-immunoprecipitates with anti-Myc in the presence of CD81-Myc and the reverse. B, endogenous TfR1 fails to interact with CD81. HEK293 cells were transiently transfected with CD81-Myc. Untransfected HEK293 cells were used as control (lane C). CD81 was immunoprecipitated with anti-Myc beads. Co-immunoprecipitated proteins were detected by Western blot with mouse anti-TfR1 and anti-Myc antibodies. C, the cytoplasmic domain of TfR2 interacts with CD81. TfR2-cyto/TfR1-tm-ecto-Flag (TfR2CD/TfR1-f) or pcDNA3 empty vector and CD81 were transiently co-transfected into HEK293 cells. TfR2CD/TfR1-f was immunoprecipitated with mouse anti-Flag antibody. Co-immunoprecipitated proteins were analyzed by immunoblot with mouse anti-Flag and mouse anti-TfR2 antibodies. D, the truncated TfR2 lacking the cytoplasmic-domain of TfR2 (TfR2ΔCD) interacts with CD81. TfR2ΔCD and CD81-Myc or empty vector were transiently co-transfected into HEK293 cells. CD81 was immunoprecipitated with mouse anti-Myc antibody. Co-immunoprecipitated proteins were analyzed by immunoblot with mouse anti-Myc and mouse monoclonal anti-TfR2 antibodies. All of the immunoprecipitations were repeated at least twice with the same results.