Cofilin is continuously activated during prolonged phagocytosis when Syk is either inhibited or its expression reduced, but transiently activated during normal phagocytosis. (A) Quantitation of phospho-cofilin-1 (p-cofilin) and total (T) cofilin after 45 min of phagocytosis by Syk-KD and Syk-inhibited (Syk-in) microglia and their respective controls (Con-Luc and Con SYK-in) based on (B) immunoblot analysis. In (A), p/T ratio in Syk-KD and Syk-inhibited microglia was calculated as percentage of p/T in their respective controls that were defined as 100 % each. Values of individual experiments, averages ± SEM, and significance of difference by double-tailed Mann-Whitney is ***p < 0.001. (C) Quantitation of p/T before (0 min) and after 10 and 30 min (min) of phagocytosis based on (D) immunoblot analysis. In (C), p/T in non-phagocytosing Con-Luc microglia was defined as 100 %. Then p/T in all other non-phagocytosing and phagocytosing microglia was calculated as percentage of p/T in non-phagocytosing control Con-Luc microglia. Average values ± SEM of three to five experiments, each performed in duplicate or triplicate, are given. Significance of differences between initial values at 0 min and those at 10 and 30 min by one-way ANOVA and the Dunnet’s post test are **p < 0.01 and ***p < 0.001 for Con-Luc microglia, and §
p < 0.05 and §§§
p < 0.001 for Syk-KD microglia. Significance of difference between Con-Luc and Syk-KD microglia by two-way ANOVA and the Bonferroni post test are #
p < 0.05 at 0 min and ###
p < 0.001 at 30 min. (E and F) Phagocytosis by Syk-KD and control Con-Luc microglia after (E) 10 min and (F) 30 min of phagocytosis. Values of individual experiments, each performed in triplicate, averages ± SEM, and levels of significance by double-tailed Mann-Whitney are given; ** p < 0.01.