Induction of Rab27a and Munc13-4 colocalization with perforin on activation of resting NK cells. Resting NK cells were incubated alone, stimulated with PMA and ionomycin, or incubated with K562 cells, as indicated, for 20 minutes at 37°C, followed by fixation, permeabilization, and labeling. Cells were labeled with anti-perforin mAb and polyclonal antibodies to Rab27a (A,E) or Munc13-4 (C,F). Confocal images of single, representative cells are shown, with scale bars representing 5 μm. Colocalization between perforin and Rab27a (B) or Munc13-4 (D) was calculated as Pearson correlation coefficient, r, using the JACOP plugin in ImageJ and averaged for 10 to 15 cells from 10 individual donors. Plots represent cumulative data from 10 to 15 cells from each of 5 donors. Plus symbols represent mean values, and error bars represent SD. Boxes represent the 25th, 50th, and 75th percentiles. Two-tailed paired Student t tests were performed to assess significance.