Rabbit monoclonal antibody against ha

COS7 cells seeded on coverslips were fixed with methanol at -20°C for 40 min, and then blocked with 5% Normal Goat Serum. HA-tagged NLGN1 was detected by mouse monoclonal antibody against HA (Cell Signaling), and ER was stained with rabbit monoclonal antibody against calnexin (Enzo). Primary neurons were fixed with 2% PFA, 4% sucrose / PBS. Rabbit monoclonal antibody against HA (Cell Signaling) and mouse monoclonal antibody against GFP (Thermo) were used. The coverslips were mounted on Vectashield mounting medium (Vector Lab). Confocal images were obtained using the laser scanning confocal microscopy (FV-1000, Olympus).

Cortex and hippocampus of mice were rapidly dissected and snap-frozen in liquid nitrogen. The tissues were homogenized in ice-cold TNE buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40), and centrifuged at 15,000 × g for 20 min at 4°C. The protein concentration was determined using the BCA protein assay (SIGMA). Cortical synaptosomes were prepared as previously described [77 (link)]. Briefly, the dissected cortex was homogenized in ice-cold HEPES-buffer (4 mM HEPES pH 7.4, 0.32 M sucrose), and centrifuged at 1,000 g for 15 min. The supernatants were subsequently centrifuged at 10,000 g for 15 min to yield the crude synaptosomal pellet. The resulting pellets were lysed with TNE buffer. Immunoreactive bands were visualized by ODYSSEY Infrared Imaging System (LI-COR), and the intensity of the band was calculated by the same system. Goat polyclonal antibody against NLGN1 (Santa Cruz) and mouse monoclonal antibody against β-actin (Sigma) were used. To detect transfected HA-tagged NLGN1 variants in cultured cells, rabbit monoclonal antibody against HA (Cell signaling) was used. Rabbit anti-NLGN1 antibody used in S3 Fig is a generous gift from Dr. Peter Scheiffele (University of Basel).