Fig. 3. MHC class II engagement induces the recruitment of I-A^k and I-A^k tr molecules into DIGs. The distribution of I-A^k and I-A^k tr molecules through the sucrose gradient and its modulation in response to I-A^k stimulation was analyzed by western blotting. (A) SaI/A^k cells were pretreated or not with MßCD for 15 minutes, and stimulated or not (nonstimulated, NS) via I-A^k for 15 minutes before lysis and fractionation on sucrose gradient. Fractions were incubated in Laemmli buffer for 30 minutes at room temperature (non-boiled) or for 10 minutes at 95°C (boiled). Samples were migrated on 10% SDS-PAGE gels, transferred to PVDF and immunoblotted with 10.2.16 mAb. Cß, compact ß dimers of MHC class II molecules; HMW, high-molecular-weight complexes; ß chain, free ß chains. (B) SaI/A^k tr cells were stimulated or not via I-A^k, lysed and fractionated as above, followed by immunoblotting with 10.2.16 mAb. (C) Immunoblots of free ß chains (boiled condition) of I-A^k and I-A^k tr were scanned and each fraction was quantified by densitometry. The results are expressed as percentage of DIG-associated MHC class II. Representative data from one of three typical experiments is shown.