Mab5220

Equal amounts of protein from cultures were subjected to 10% SDS-polyacrylamide gels and transferred to PDVF membranes (Millipore). The blots were blocked with 5% nonfat dry milk or 3% bovine serum albumin (BSA) for 1 h at room temperature followed by incubation with various primary antibodies, including GluA2 (1:500, MAB397, Chemicon), GluN1 (1:500, MAB363, Chemicon), GluN2B (1:500, MAB5220, Chemicon), rabbit anti-GABA_A α1 receptor (1:500, AGA001, Alomone Labs), synapsin 1 (1:10000, AB1543, Chemicon), GAPDH (1:10000, AB2302, Millipore), and anti-Phospho-Tyr1472 NMDA GluN2B-Subunit (1:500, AB5403, Chemicon). After incubation with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences), the blots were exposed to the enhanced chemiluminescence substrate (Amersham Biosciences). Quantitation was obtained from densitometric measurements of immunoreactive bands on films with Image J software.

After nicotine treatment, neurons in culture were fixed with 2% paraformaldehyde in PBS with 4% sucrose (RT, 10 min), or with 100% methanol (−20°C, 5 min) and washed 3 times with PBS. For total protein staining, neurons were permeabilized with 0.1% Triton X-100 in PBS for 10 min. For surface protein staining, neurons were not permeabilized. Neurons were incubated with 5% BSA for 1 h to block nonspecific staining. Next, neurons were incubated with primary antibodies at 4°C overnight, including GluA2 (1:200, MAB397, Chemicon), GluN1 (1:200, MAB363, Chemicon), GluN2B (1:200, MAB5220, Chemicon), or synapsin 1 (1:800, AB1543, Chemicon) overnight at 4°C. Neurons were then rinsed in PBS three times and exposed to Alexa-conjugated fluorescent secondary antibodies for 2 h (Molecular Probes, 1:1000) at room temperature. After washing in PBS three times, the coverslips were mounted on slides with VECTASHIELD mounting media (Vector Laboratories, Burlingame, CA).