Figure 6.

C5b-9 deposition triggers mixed lineage kinase domain-like protein (MLKL) translocation to the plasma membrane and C5b-9-MLKL interaction. (A) K562 cells were treated with a sublytic dose of antibody and with complement [normal human serum (NHS)] or HIS for 10 min at 37°C. After fixation and permeabilization, MLKL was stained with anti-MLKL antibody and AF546-labeled secondary antibody. Representative confocal microscope images are presented. NT, non-treated cells. (B) K562 cells were treated with antibody and complement as above. In addition to MLKL labeling, C5b-9 was detected with an anti-C5b-9 antibody (AE-11) and with AF488-labeled secondary antibody. MLKL and C5b-9 locations were merged by the Co-localization plugin in ImageJ software. Representative cells (from three independent experiments) show the co-localization of MLKL with C5b-9 in the plasma membrane region. (C–E) K562 cells were treated with complement (NHS) as above and cell lysates were prepared. Controls included HIS and C8-depleted human serum (C8D). (C) Cell lysates were subjected to immunoprecipitation with anti-C9 antibody followed by Western blot analysis with anti-C9 or anti-MLKL antibodies. (D) MLKL was immunoprecipitated from cell lysates with an anti-MLKL antibody. MLKL was detected by Western blotting. MLKL and actin were also quantified in whole-cell lysates (Lysates). (E) Proteins immunoprecipitated from cell lysates with anti-MLKL antibodies, as in (D), were eluted from the beads and the amount of C5b-9 was quantified by ELISA. Results are expressed as adsorption at 450 nm (A450). *P < 0.05 relative to C8D or HIS treated cells (t-test).