Fig. 6. Truncation of I-A^k prevents DIG-dependent PKC- translocation to the site of I-A^k engagement. (A) SaI/A^k and SaI/A^k tr cells were incubated with 10.2.16-coated beads (Anti-I-A^k) or irrelevant mouse Ig-coated beads (nonstimulated, NS) for 30 minutes. Cells were cytospun, fixed and stained with anti-PKC- antibody and revealed with a FITC-labeled secondary antibody. Bright-field images (a,c,e,g,i,k) indicate the site of interaction between the bead and the cell and the corresponding fluorescent images reveal the localization of PKC- (b,d,f,h,j,l). A single cell-bead conjugate is shown in each panel. Cells pretreated with MßCD prior to I-A^k stimulation (Anti-I-A^k + MßCD) are indicated. (B) The percentage of cells undergoing PKC- recruitment to the site of I-A^k engagement was determined. At least 200 cell-bead interactions from each condition were examined and cells in which PKC- recruitment had occurred (to at least the same degree as shown in A, panel d) were counted as positive events. Data from one of three typical experiments is shown.