Mmp14

Western blot analysis was performed as described previously (Nizzardo et al., 2014a (link); Rizzo et al., 2016 (link)). Briefly, cells were sonicated on ice for 10 min in buffer supplemented with protease and phosphatase inhibitor cocktail (Pierce) (Nizzardo et al., 2014a (link); Rizzo et al., 2016 (link)). Alternatively, 20 mg of frozen brain were homogenized in 0.4 ml of protein sample buffer containing 2% (w/v) SDS, 10% (v/v) glycerol, 50 mM Tris-HCl (pH 6.8), and 0.1 M DTT. A total of 50 µg was separated via 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Proteins were transferred to a nitrocellulose membrane and incubated with primary antibodies overnight at 4°C. The primary antibodies used in these experiments were: NFs (1:1000, Sigma), MMP14 (Thermofisher, 1:1000), SYT13 (1:1300, Proteintech), NRXN1 (1:2000, Abcam), NRXN2α (1:500, Abcam), SMN (1:1.000, BD), STMN2 (1:2000, Proteintech), PLP1(1:800, Sigma), and hnRNP-Q (1:1000, Sigma). The blots were then incubated in secondary antibody: polyclonal anti-rabbit (1:2700, Dako) and polyclonal anti-mouse (1:3200, Dako). The immune complexes were revealed by chemiluminescence assay (Amersham). The nitrocellulose membrane was stripped and reprobed with anti-actin (1:1000, Sigma) as a loading control. Densitometry was performed using ImageJ software.