FIGURE 3.

CD9 is not essential for the association of MHC-II I-A and I-E molecules. A, MHC-II molecules were immunoprecipitated (IP) from Brij-58 (B) or Triton X-100 (T) lysates of activated DCs using isotype control, I-A-, or I-E-specific mAb and analyzed by immunoblotting using I-E α-chain, I-A β-chain, or CD9 antibodies as indicated. An aliquot of the cell lysate was also analyzed. A representative gel is shown. B, LPS-activated DCs were prepared from wild-type (WT), CD9 heterozygous (Het), and CD9-deficient (KO) mice crossed onto the H-2^k background and lysed in Brij-58 lysis buffer, and CD9 expression was determined by immunoblotting. C, DCs were prepared from CD9 heterozygous (control), and CD9-deficient mice crossed onto the H-2^k background. MHC-II molecules were immunoprecipitated from Brij-58 lysates of activated DCs using I-A or I-E-specific mAb and analyzed by immunoblotting as indicated. An aliquot of the cell lysate was also analyzed. The relative amount of I-A or I-E present in each immunoprecipitate was determined by densitometry and expressed as a fraction of the amount of I-A or I-E present in the cell lysate, and the binding of I-E to I-A and I-A to I-E in CD9 KO DCs was normalized to that obtained in the heterozygous controls. The mean ± S.D. from three independent experiments is shown.