Synaptophysin

GST-fusion protein containing human Neph2 (aa 563–778) and synthetic peptide mimicking the last 10 aa of human Neph2 were used to immunize rabbits (1344 and 1468, respectively). For CaMKIIα/β polyclonal antibodies, GST-fusion proteins containing full-length CaMKIIα were used to immunize guinea pigs (Gp). The following antibodies have been described: EGFP (1173, Rb; Ko et al., 2003 (link)), PSD-95 (1402, Gp), SAP102 (1447, Gp) (Choi et al., 2005 (link)), PSD-93 (1634, Rb), SAP97 (1443, Gp) (Oh et al., 2010 (link)), CASK (1640, Rb), GluA1 (1193, Rb), GluA2 (1195, Rb) (Kim et al., 2009 (link)). The following antibodies were purchased: synapsin I (Chemicon), synaptophysin (Santa Cruz), GluN2A (Invitrogen), GluN2B (BD Transduction Lab), mGluR5 (Millipore), PAK1/3, p-PAK1/3, LIMK1, p-LIMK1, Cofilin, p-Cofilin, and ROCK1 (Cell Signaling), α-Tubulin (Sigma), and NeuN (Millipore).

Protein concentrations of samples were determined using a BCA protein assay kit (Thermo Fisher Scientific) and Ponceau Red staining of membranes. Equal protein amounts were electophoresed on 10% tris-glycine SDS-polyacrylamide gels, Nu-Page 4–12% or 10% bis–tris gels (Invitrogen), transferred to 0.45-μm nitrocellulose membrane (Millipore, MA, USA) and immunoblotted using standard methods. Primary antibodies were BIN1 (99D; Millipore), GST (GE Healthcare, IL, USA), total tau (total human tau; Agilent), Tau-1 (Millipore), PHF1 (Peter Davies, Donald and Barbara Zucker School of Medicine at Hofstra, Northwell), N-methyl-d-aspartate subunit 2B (06-600; Millipore), β-actin (ac15; Abcam), synaptophysin (sc17750; Santa Cruz), PSD95 (MAB 1596; Millipore) and Fyn (HPA023887; Sigma). The bound horseradish peroxidase-conjugated secondary antibodies (GE Healthcare) were detected using enhanced chemiluminesence solutions (Thermo Fisher Scientific) and visualized using a ChemiDoc imager (Bio-Rad, CA, USA). Densitometric analysis was performed using FIJI.

Protein lysates from BV2 cells, N2a cells, BMDMs and brain tissue were harvested in RIPA buffer, supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich, UK). Proteins (5, 10 or 20 µg) were separated using SDS-PAGE, transferred to nitrocellulose membranes and blocked in 5% semi-skimmed milk prior to overnight incubation with antibodies against iNOS (1:1,000; BD Biosciences, UK), nNOS (1:500 or 1: 1000; Cell Signaling, UK), COX2 (1:1000), PSD-99 (1:1000; Santa Cruz, USA), drebrin (1:1000; Santa Cruz, USA), synaptophysin (1:1000; Santa Cruz, USA) and β-actin (1:2,000; Santa Cruz, USA). Membranes were washed and incubated with DyLightTM 680/800 fluorescent anti-mouse or anti-rabbit secondary antibodies (Thermo Scientific, UK). Fluorescent immunoreactive bands were visualised using the LI-COR Odyssey and quantified with Image Studio Lite software.

Peptides containing mouse Clmp (aa 345–373) were used to immunize guinea pigs (2090). The specificity of anti-Clmp antibodies (2090) was confirmed by immunoblot experiments using Clmp^−/− whole-brain lysates. The following antibodies have been described: PSD-95 (1688) (Yang, 2011 (link)), GluA1 (1193) (Kim, 2009 (link)), GluA2 (1195) (Kim, 2009 (link)). The following antibodies were purchased: HA rabbit polyclonal (Santa Cruz sc-805), HA mouse monoclonal (Boehringer Mannheim 12CA5), PSD-95 (75-028) (NeuroMab), Synaptophysin (Santa Cruz sc-9116), GluA1 (Sigma-Aldrich MAB2263), GluA2 (Sigma-Aldrich MAB397), GluK2 (Sigma-Aldrich 04-921), GluK5 (Sigma-Aldrich 06-315), α-tubulin (Sigma T5168), β-actin (Sigma, A5316).

Antibodies against GFP (Rockland), S100 (Dako), synaptophysin (Santa Cruz), neurofilament (Millipore) and acetylcholinesterase (kindly provided by P. Taylor, UCSD) were used at 1/1000 in PBS containing 1% triton-X and 10% fetal bovine serum to detect proteins in fixed, whole-mount diaphragms. Fluorescently-conjugated α-BTX and fasciculin-2 were added with secondary antibodies. Tissues were confocally imaged with an Olympus Fluoview 1000. For myosin heavy chain staining, muscles were fresh-frozen, cut at 16 μm, and immediately incubated without fixation in PBS with primary antibodies as described (Heredia et al., 2016 (link)).