Af6379

C5–C7 spinal segments or musculocutaneous nerve were cut into 15-µm frozen sections for immunostaining. The blocking buffer was composed of 5% goat serum and 3% bovine serum albumin diluted in 0.1 M PBS. Signal was detected with Alexa Fluor 546 or 488 coupled secondary antibodies (1:1000, Invitrogen). Primary antibodies were: goat anti-ChAT (1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Isl1 (1:1000, Abcam) and goat anti-human CELSR2 (1:100, AF6379, R&D systems). To visualize neuromuscular junctions (NMJs), the biceps were collected 50 days post-surgery and the wet weight was recorded. Seventy-micrometre horizontal sections were prepared with a sliding microtome (Leica, Germany) and double stained with rabbit anti-NF200 (1:500, n4142, Sigma) and α-BT (1:1000, Molecular probes). To analyse axon number in musculocutaneous nerves, tissues were fixed with 2.5% glutaraldehyde plus 2% paraformaldehyde and 500-nm semi-thin sections were stained with 1% Toluidine Blue; images were taken under a 63× oil objective.

Protein extracts from E13.5 mouse cervical segments, C5–C7 ventral horns of adult animals 3 days after surgery, 5-DIV cultured human motor neuron explants and proteins eluted from glutathione-agarose beads were analysed on 10% SDS polyacrylamide gels and then transferred to 0.45-μm nitrocellulose membranes. The following primary antibodies were used: anti-β-tubulin (Tuj1) rabbit polyclonal antibody (1:1000; ab18207, Abcam), anti-GAPDH mouse polyclonal antibody (1:1000; ab8245, Abcam), anti-JNK rabbit polyclonal antibody (1:1000; 9252, CST), anti-c-Jun rabbit polyclonal antibody (1:1000; 3270S, CST), anti-EB3 rat polyclonal antibody (1:1000; ab53360, Abcam), anti-GSK-3β polyclonal antibody (1:1000; 9315, CST), anti-Rac1 mouse polyclonal antibody (1:1000; 610650, BD Pharmingen), anti-CDC42 rabbit polyclonal antibody (1:1000; 11A11, CST), goat anti-human CELSR2 (1:1000, AF6379, R&D systems); the secondary antibodies include peroxidase anti-rabbit IgG (1:5000, ab6721, Abcam) and peroxidase anti-mouse IgG (1:10 000; Vector Laboratories). Immunoreactivity was detected using an enhanced chemiluminescence detection kit (1705061, Bio-Rad).