Fig. 5. Loss of Nck protein in the nervous system leads to developmental defects. (a) Southern blot of genomic DNA isolated and digested with HindIII, from litters of animals generated by injecting fertilized eggs from a cross between an Nck1^+/+;Nck2^flx/- female and an Nck1^+/+;Nck2^flx/+ male with a vector expressing Flp recombinase or Cre recombinase. Note the successful excision of sequence between the loxP sites in the presence of a Cre vector (from 10.2 kb to 7.3 kb) (lane1 vs. lane 2), and the successful excision of the sequence between the flip sites (from 10.2 kb to 8.0 kb) (lane 4 vs. lane 5) in the presence of Flp vector. Lane 3 shows wild-type alleles for Nck2 (8.0 kb). (b) Mouse embryonic fibroblasts (MEFs) generated from Nck1^+/-;Nck2^flx/- and Nck1^-/-;Nck2^flx/- mice were treated with or without an adenovirus expressing Cre recombinase. At 48 h after infection, cells were harvested, equal amounts of protein were loaded, and Nck expression was determined using a pan-Nck antibody. (c) Typical example of genotypes obtained from crossing a Nck1^-/-;Nck2^flx/flx female with a Nck1^-/-;Nck2^flx/-;Nestin-Cre male. (d) Comparison of the fold change in Nck2 mRNA levels from the cortex of individual P1 animals relative to a Nck1^-/-;Nck2^flx/-;Nestin-Cre littermate. Gene expression was measured by quantitative RT-PCR using cyclophilin-A as an internal control. (e) Fold change in Nck2 expression in a series of Nck1^-/-;Nck2^flx/flx;Nestin-Cre-positive animals in d relative to a Nck1^-/-;Nck2^flx/-;Nestin- Cre littermate. Results were similar for two independent sets of Nck2 primers. (f) P12 animals showing two littermates. The genotype of the smaller animal was Nck1^-/-;Nck2^flx/-;Nestin-Cre.

Fig. 6. Loss of Nck protein in the nervous system does not alter EphA4 expression or ephrin-B3 localization (a and b) Lysates generated from the lumbar spinal cord (a) or brains (b) of adult animals from various genotypes were probed for Nck (Top), EphA4 (Middle), or tubulin (Bottom). (c) Immunoblotting of brain lysates with an ephrin-B3 specific antibody demonstrates the specificity of the ephrin-B3 antibody. (d and e) Coronal sections through the thoracic region of spinal cords from P2 Nck-deficient (Nck^-/-;Nck2^flx/flx;Nestin-Cre) (d) or a littermate control (Nck1^+/-;Nck2^flx/flx) (e) stained with the ephrin-B3 antibody.

Movie 1. Movie documents the locomotion pattern of a postnatal day 4 (P4) control and a Nck-deficient (Nck1^-/-;Nck2^flx/flx;Nestin-Cre) mouse. Note the symmetric movement of the left and right limbs associated with the Nck-deficient mutants.

Movie 2. Movie documents the locomotion pattern of a postnatal day 12 (P4) Nck1^-/-;Nck2^flx/-;Nestin-Cre mouse. Note the flexion/extension seizure-like activity of the hind limbs in response to stimulation.