Figure 4. Netrin-1–induced Pak1-mediated shootin1a phosphorylation enhances the interaction between shootin1a and L1-CAM.

(A and B) Co-immunoprecipitation of myc-shootin1a and FLAG-L1-CAM-ICD in HEK293T cells. Cells were transfected with vectors to express myc-shootin1a and FLAG-L1-CAM-ICD; some of them were also co-transfected with a vector to express dominant negative Pak1 (KD) or constitutively active Pak1 (CA) as indicated. Cell lysates were then incubated with anti-FLAG antibody. The immunoprecipitates were immunoblotted with anti-myc or anti-FLAG antibody (A). Cell lysates (1%) were also analyzed with anti-pSer101-shootin1, anti-pSer249-shootin1, or anti-myc antibody. Quantitative data for phosphorylated and bound shootin1a are shown in (B) (n = 3 independent experiments). Data represent means ± SEM; ***p<0.01; **p<0.02; *p<0.05 (One-way ANOVA with Tukey’s post hoc test). (C and D) Co-immunoprecipitation of shootin1a and L1-CAM in cultured cortical neurons. After incubation of neurons with 4.4 nM netrin-1 or BSA (control) for 1 hr, cell lysates were prepared and incubated with anti-shootin1 antibody (right panel). The immunoprecipitates were immunoblotted with anti-shootin1 or anti-L1-CAM antibody. The cell lysates (5%) were also analyzed with anti-pSer101-shootin1, anti-pSer249-shootin1, or anti-shootin1a antibody (left panel). Quantitative data for phosphorylated shootin1a and bound L1-CAM are shown in (D) (n = 3 independent experiments). Data represent means ± SEM; **p<0.02; *p<0.05 (Unpaired Student’s t-test). (E) Fluorescence images of an axonal growth cone labeled with anti-pSer249-shootin1a (magenta) and anti-L1-CAM (green) antibodies. The cells were observed using a TIRF microscope. An enlarged view of the filopodium in the rectangle is shown in the lower panel. Arrowheads indicate phosphorylated shootin1a colocalized with L1-CAM. Bar: 5 μm (in the inset, 2 μm).