Fig. 8. Recruitment of MHC class II molecules to DIGs is independent of MHC class II-mediated signaling. (A) SaI/A^k cells were pretreated with the inhibitor of actin polymerization cytochalasin D (Cyt.D), or with the PKC inhibitor calphostin C (Calph.C) prior to engagement of I-A^k for 15 minutes at 37°C. I-A^k recruitment to the DIG-containing fraction isolated from SaI/A^k was unchanged in the presence of the above inhibitors as detected by immunoblotting. (B) The effect of cytochalasin D on actin reorganization induced by I-A^k engagement was revealed by confocal microscopy of cell-bead conjugates. Panels a and b represent, respectively, bright-field imaging and Alexa488-phalloidin staining of SaI/A^k cells stimulated with 10.2.16-coated beads. Panels c and d represent, respectively, bright-field imaging and Alexa488-phalloidin staining of SaI/A^k cells pretreated with Cyt.D prior to 10.2.16-coated beads stimulation. (C) The effect of calphostin C on PKC- recruitment induced by I-A^k engagement was revealed by confocal microscopy of cell-bead conjugates. Panels a and b represent, respectively, bright-field imaging and PKC- staining of SaI/A^k cells stimulated with 10.2.16-coated beads. Panels c and d represent, respectively, bright-field imaging and PKC- staining of SaI/A^k cells pretreated with Calph.C prior to 10.2.16-coated beads stimulation.