Figure 3: Developmental expression pattern of mLLP and its roles in the dendritic morphogenesis.

(A) Western blot analysis of the whole brain lysates of mice from embryonic stages to adult. mLLP protein level in the brain is gradually decreased during development. Loading controls (β-tubulin) were detected on the same blot. Samples are derived from the same experiment and processed in parallel. (B) Western blot analysis of mLLP expression level in dissociated mouse hippocampal neuron cultures with time. Loading controls (GAPDH) were detected on the same blot. Samples are derived from the same experiment and processed in parallel. (C) Representative microscopy images of neurons at DIV 6 transfected differently into following groups at DIV 3: control (CTL, scrambled shRNA + control vector), mLLP overexpression (OE, scrambled shRNA + shRNA-resistant mLLP), mLLP knockdown (KD, shRNA against mLLP + control vector), or mLLP rescue (RES, shRNA against mLLP + shRNA-resistant mLLP). Scale bar, 50 μm. (D) Sholl analysis of neurons transfected with constructs for KD, RES, or CTL (two-way ANOVA, n = 24–25 cells per group, interaction ***p < 0.0001, group effect ***p < 0.0001). (E) The ratio of primary dendrites shorter than 30 μm (one-way ANOVA and post hoc Tukey’s multiple comparison test, *p < 0.05, ***p < 0.001, n = 29–30 cells per group). (F) Sholl analysis of mLLP overexpressing neurons or control neurons (n = 30 cells per group, two-way ANOVA, group effect **p = 0.0048). (G) Sholl analysis at DIV 6 after daily treatment with mLLP (1μg/mL) or control GFP protein for 3 days (n = 29 cells per group). Extracellular addition of mLLP protein increased the dendritic arborization compared with control. (two-way ANOVA, group effect *p = 0.0338). All the data are represented as mean ± SEM.