Myosin II inhibition does not affect Arp2/3 complex localization or barbed-end distribution. (A) Fluorescent labeling of growth cones with Arp3 antibody (right) and Alexa 594 phalloidin (left) after normal fixation. Growth cones were treated with blebbistatin (60 µM, 20 min, top) or pretreated with blebbistatin (60 µM, 10 min) followed by blebbistatin and CK666 (100 µM, 20 min, bottom). (B) Arp3 distribution profile sampled from the designated lines in A. (C) Quantification of Arp2/3 complex enrichment at the leading edge for each condition in A. The control from Fig. 1 E is shown for comparison. Numbers in parentheses indicate growth cones measured. *, P < 0.01 with two-tailed unpaired t test. (D) Growth cones were dual-labeled with TRITC-phalloidin to show total F-actin (left) and Alexa 488 G-actin, which incorporates at barbed ends (right). Growth cones were treated with blebbistatin (60 µM, 20 min, top) or pretreated with blebbistatin (60 µM, 10 min) followed by blebbistatin and CK666 (50 µM, 20 min, bottom). Yellow dotted line demarcates leading edge. Arrowheads: filopodia. (E) Line scan analysis of barbed end localization in growth cones under each condition in D. Scattered dots represent dataset from individual growth cones. Solid lines represent the population average. n, growth cones measured. (F) Average barbed-end intensities in the distal half of the P-domain in each condition. Data for control and CK666 (50 µM, 20 min) from Fig. 3 C are shown for comparison. *, P < 0.01 with two-tailed unpaired t test. NS, not significant. Bars: (A) 10 µm; (D) 5 µm.