FIGURE 2.

Developmental regulation of MHC-II I-A/I-E interactions in DCs. A, untreated or LPS-activated DCs were lysed in 1% Brij-58 lysis buffer, and I-A or I-E was immunoprecipitated (IP) using specific mAb, and the immunoprecipitates were analyzed by immunoblotting using the indicated antibodies. Aliquots of each lysate were also loaded on the gels. B, cell surface proteins of untreated or LPS-activated DCs were surface biotin-labeled on ice; the cells were washed and finally lysed in 1% Brij-58 lysis buffer. Lysates were depleted of I-A or I-E MHC-II molecules using 10.2.16 or 14.4.4S mAb-coated protein A-Sepharose beads. Residual surface proteins were isolated from these immunodepleted lysates using streptavidin-agarose beads and analyzed by immunoblotting by using the indicated antibodies. A representative gel indicating the amount of biotinylated (surface) I-E and I-A MHC-II remaining after the control, I-A, or I-E specific-immunodepletion is shown. C, intensity of each band in B was determined by quantitative densitometry. The amount of surface I-E remaining in the I-A immunodepleted lysate (i.e. free) and I-A remaining in the I-E immunodepleted lysate (i.e. free) was expressed as a percentage of amount of surface I-A or I-E remaining in the control immunodepleted lysates. The percentage of total surface I-E bound to I-A or I-A bound to I-E was calculated as 100% − %free. The mean ± S.D. from three independent experiments is shown.