Fig. 3

Requirement of ARF1 in RAC1 activity and its translocation to the leading edges. (a and b) Activity of ARFs in differentiated HL-60 cells after fMLP stimulation. Activities of class I and II ARFs were measured by GST-GGA3 pulldown coupled with the indicated antibodies. Each lower panel represents immunoblots of the total cell lysates (5 μg) by the indicated antibodies. Data are representative of three independent experiments (a), and were analyzed in three independent experiments (b). In b, values of each GTP-ARF at 0 s are considered 1. ** p < 0.01 and * p < 0.05 compared with each GTP-ARF at 0 s. (c and d) Subcellular localization of RAC1, ARF1, and ARF5 after fMLP stimulation. Differentiated HL-60 cells were incubated with fMLP for 5 min, and subjected to immunostaining analysis, using high-resolution SIM. Specificities of the anti-ARF1 antibody and the anti-RAC1 antibody were confirmed by ARF1 or RAC1 siRNA-treatment of cells (c) Bars, 2 μm. Pearson’s correlation coefficients of the intracellular colocalization of these proteins, as indicated, were estimated from >10 cells (d). (e) Suppression of ARF1 or ARF5 by siRNAs in differentiated HL-60 cells. Cells transfected with siRNA against ARF1, ARF5, or an irrelevant RNA duplex (Irr) were analyzed for the expression of the indicated proteins by immunoblotting of the lysates (10 μg each). Data are representative of three independent experiments. (f and g) Subcellular localization of RAC1. Differentiated HL-60 cells, transfected with siRNA against ARF1, ARF5, or Irr, were incubated with fMLP for 15 min, and subjected to anti-RAC1 immunostaining (f), and percentages of RAC1 molecules translocated to the leading edges in fMLP-stimulated cells were calculated (g). (h and i) Activities of RAC1. Activities of RAC1 were measured by GST-PBD pulldown coupled with the anti-RAC1 antibodies. Each lower panel represents immunoblots of total cell lysates (5 μg) by the anti-RAC1 antibodies. Data are representative of three independent experiments (h), and were analyzed in three independent experiments (i). In i, values for Irr control at 0 s are considered 1. (j) Suppression of RAC1 by siRNAs in differentiated HL-60 cells. Cells transfected with siRNA against RAC1 or Irr were analyzed for expression of the indicated proteins, by immunoblotting of the lysates (10 μg each). Data are representative of three independent experiments. (k and l) Subcellular localization of ARF1. Differentiated HL-60 cells, transfected with RAC1 siRNA or Irr, were incubated with or without fMLP, as indicated, and subjected to anti-ARF1 immunostaining (k), and percentages of ARF1 molecules translocated to the leading edges in fMLP-stimulated cells were calculated (l). F-actin was visualized by Texas Red-phalloidin. Data are representative images of three independent experiments (f, and k), and >25 cells were analyzed in three independent experiments (g and l). Error bars, SEM (b, d, g, i and l). ** represents a statistical difference from Irr (p < 0.01) (g and i). Bars, 10 μm (f and k)