Defective MTOC polarization in AKAP450 knockdown cells. J77 cells (A) and CH7C17 cells (B) were transfected with a control siRNA or AKAP450 siRNA, and cell conjugates were formed as in Figure 5A. Conjugates were stained for endogenous AKAP450 (red) and α-tubulin (green). Yellow arrows indicate the positions of immune synapses; blue arrows, position of the mislocalized MTOC in AKAP450 knockdown cells. CMAC-labeled SEE-pulsed Raji and HA-pulsed HOM-2 APCs are distinguished by cyan fluorescence in DIC images (*). Scale bars represent 10 μm. (A-B) Histograms show quantification of MTOC translocation in (A) J77 and (B) CH7C17 cells. More than 200 conjugates were counted for each condition. Results are the arithmetic mean ± SD of MTOC translocation. Control incubation with uploaded APCs represented as −SEE and −HA in the graphs. *P < .05 compared with T cells transfected with control siRNA (Student t test).