Western Blotting

E13.5 MGE tissue was dissected and lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate in 50 mM Tris) supplemented with protease (Halt protease inhibitor, Pierce) and phosphatase (PhosSTOP, Roche) inhibitors. ~20 μg of total protein was loaded into an SDS-PAGE gel, separated, and transferred to a nitrocellulose membrane. The membrane was probed with the following antibodies: rabbit anti-CASPR2 (Millipore), rabbit anti-pAKT^Ser473 (Cell Signaling), rabbit anti-total AKT (Cell Signaling), pERK1/2^{threonine 202/tyrosine 204} (Cell Signaling), total ERK1/2 (Cell Signaling), mouse anti-βIII-tubulin (Covance), and the species-appropriate HRP-conjugated secondary antibodies (Biorad). HEK293T cells were transfected with human CNTNAP2 expression vectors using Lipofectamine²⁰⁰⁰ (ThermoFisher), and cell lysates were collected after 48 h in the same manner described above. The rabbit anti-CASPR2 (Millipore), rabbit anti-GFP (ThermoFisher) and appropriate HRP-conjugated antibodies (BioRad) were used to detect proteins.