Fig. 4. MT81w only binds to CD81 associated with tetraspanins. (A) Hepa1-6 cells were lysed in the presence of CHAPS, Brij 97, digitonin or Triton X-100 before immunoprecipitation with the anti-CD81 mAbs MT81 and MT81w or with the anti-CD9 mAb 4.1F12. Immunoprecipitates were analyzed by western blotting using biotin-labeled anti-CD81 (MT81) and anti-CD9 (4.1F12) mAbs. Note that MT81w precipitates both CD81 and CD9 in CHAPS and Brij 97, but not in digitonin or Triton X-100. (B) Hepa1-6 cells were lysed in 1% Brij 97 before five rounds of immunoprecipitation with anti-CD9 mAb 4.1F12 to deplete CD9 (depl. +) or with beads alone as a control (depl. -). The supernatants were then used for immunoprecipitations as in A. The membranes in each panel were given the same exposure.