GAPDH is necessary for axonal retraction. (A) Endogenous GAPDH expression was detected by immunostaining (in green) at the growth cone and adjacent axonal shaft in hippocampal neurons cotransfected with siRNAs and LifeAct-mCherry (in magenta) for F-actin visualization and as a transfection control. The outline of the growth cones was drawn (white dashed lines) on the LifeAct-mCherry channel. (B) Boxplot showing GAPDH intensity at individual growth cones (GC). Mean intensity of GAPDH was measured on the region defined by LifeAct-mCherry fluorescence, as shown in A; 36 < n < 55 axons from two independent experiments. (C) WIN (100 nM)-induced axonal retraction assay showing the average distance from position at the beginning of the experiment. 36 < n < 97 axons from three independent experiments. (D and E) Boxplot showing the distance of the growth cone at 20 min after WIN induction as well as after 30 min growth; 36 < n < 97 axons from three independent experiments Statistics: one-way ANOVA followed by Tukey honestly significant difference (HSD) post-test correction; ***P < 0.001.