Seine threonine phosphatase inhibitor

Cultured rat hippocampal neurons at DIV14–16 were lysed with 1% triton X-100 lysis buffer with 1% seine/threonine phosphatase inhibitor (Sigma) and protease inhibitor cocktail (Sigma). Lysates were centrifuged at 14,000 g at 4 °C for 20 min after sonication. Supernatants were collected and protein concentration was measured using BCA assay kit. Equal amounts of protein were loaded on to polyacrylamide gels. Gels were transferred to PVDF membranes (Pall Life Sciences, Ann Arbor, MI), then the membranes were incubated with 10% BSA/PBS or 5% SKIM milk/PBS for 30 min at RT. After washing in TBST, PVDF membranes were incubated with the primary antibody for overnight at 4 °C, followed by the horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at RT. ECL solution (AbClon) and LAS 4000 (GE healthcare) were used to detect immunoreaction. Band intensities were calculated using imageJ.

Transfected HEK293T cells were lysed in a 1% triton X-100 lysis buffer [20 mM Tris-HCl, pH 8, 1% triton X-100, 10% glycerol, 137 mM NaCl, 2 mM EDTA] with 1% seine/threonine phosphatase inhibitor (Sigma) and protease inhibitor cocktail (Sigma). After sonication and centrifugation, anti-synapsin I antibody (Synaptic Systems) was added to each of equal amounts of total cell lysate (500 μg). The samples were incubated overnight at 4 °C, then 30 μl Protein A-Sepharose (GE healthcare) was added and incubated for 1 h at 4 °C. The samples were washed three times with lysis buffer and then bead pellets were eluted with 30 μl of 2× sample buffer [100 mM Tris-HCl (pH 6.8), 4% sodium dodecyl sulfate, 0.2% bromophenol blue, 20% glycerol and 2% beta-mercaptoethanol] followed by boiling (100 °C, 5 min) and gel loading.