Fig. 4. Effect of siRNA expression on the number of vesicles docked to the plasma membrane in PC12 cells. (A) Typical TIRF images of plasma membrane-docked vesicles before high-KCl stimulation of control (a), Rab3A-siRNA-expressing (b), Rab27A-siRNA-expressing (c) and Rab33A-siRNA-expressing cells (d). (e-h) Magnified images of the boxed area. Bars, 5 µm. (B) The density of docked vesicles was determined by counting the vesicles in each image (n=6 cells in each). The inset shows NPY-Venus and actin protein expression visualized with anti-GFP and anti-actin antibody, respectively. The positions of the molecular mass markers (x10^-3) are shown on the left. (C) The number of NPY-Venus spot disappearance events was counted as fusion events in a 5-minute period (n=6 cells in each). The data are mean values ± s.e.m. and were analyzed by one-way ANOVA followed by Newman-Keuls multiple comparison test. ^*P<0.05 and ^**P<0.01, respectively, compared with the control. Note that expression of either Rab3A or Rab27A siRNA significantly reduced the number of plasma membrane-docked vesicles (B) as well as the number of NPY-Venus release events (C).