Figure S4. Effect of Nrg1 loss on axonal growth pathways and rescue by GAP43.
(A) Representative images of Western blot (WB) analysis showing the expression levels of total and phosphorylated AKT, JNK, and ERK proteins in control (Nrg1flox/flox) and Nrg1-deficient (Nes-Cre; Nrg1flox/flox) neurons at DIV4. Tubulin levels are shown as a loading control. (B) Quantification of total and phosphorylated AKT, JNK, and ERK protein levels from the WB analysis, normalized to tubulin. Ctrl: n = 2; Nrg1 KO: n = 3, littermates. Unpaired t tests, in all cases, P > 0.29. Data are presented as the mean ± SEM. (C) Representative images and schematic drawings of control, Nrg1-deficient (Nrg1 KO), and Nrg1 KO neurons expressing GAP43, including the drawings used for Sholl analysis of the dendrites. Cells were fixed at DIV4. Scale bar, 50 μm. (D) Graphs showing the quantification of dendrite length in control, Nrg1 KO, and Nrg1 KO neurons expressing GAP43, based on Sholl analysis. Data are presented as the mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparisons test was used to compare Ctrl versus Nrg1 KO– and GAP43-expressing Nrg1 KO neurons. Ctrl: n = 45; Nrg1 KO: n = 44; Nrg1 KO+GAP43: n = 37, individual neurons from two different experiments. ns, not significant. (E) Quantification of axonal length in control, Nrg1 KO, and Nrg1 KO neurons expressing GAP43, expressed in microns. Ctrl, n = 104: Nrg1 KO: n = 105; GAP43: n = 78, neurons from two independent experiments. Data are presented as the mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparisons test, **P < 0.001, ***P < 0.001.