Reagents and antibodies
Active Abl kinase (mouse) was purchased from Upstate Biotechnology/Millipore (Belerica, MA). Each lot of active Abl kinase was tested for phosphorylation of a peptide substrate in an in vitro kinase assay (by the manufacturer). Briefly, each lot of Abl kinase had a specific activity (~1750 units/mg) where 1 unit is defined as incorporating 1 nmol phosphate into 50 μM Abltide (EAIYAAPFAKKK) per minute at 30°C in the presence of 100 μM ATP. PDGF-BB was purchased from R&D Systems (Minneapolis, MN, USA). Y-27632 was purchased from Calbiochem (San Diego, CA). NMDA, glycine, and all other chemical reagents were purchased from Sigma (St. Louis, MO, USA). Antibodies used include those raised against PDGFβ receptor (Epitomics, California, USA), Abelson kinase (Sigma), Src (Cell Signaling, Danvers, MDA) and phospho-Y416 and Y527 Src (Biosource, Carlsbad, CA), and phospho-tyrosine antibody (Santa Cruz, La Jolla, CA), and PLCγ, ROCK, and WAV1 (Cell signaling). For the immunoprecipitation of PDGFβ receptors, an agarose-conjugated anti-PDGFβR antibody from Santa Cruz was employed.
Cell isolation and whole-cell recording
CA1 neurons were isolated from hippocampal slices of postnatal day 14–21 Wistar rats as previously described . The extracellular solution was composed of 140 mM NaCl, 1.3 mM CaCl2, 25 mM N-2-Hydroxyethylpiperazine-N'-thanesulfonic acid (HEPES), 33 mM glucose, 5.4 mM KCl, and 0.5 μM tetrodotoxin, and 0.5 μM glycine, with pH of 7.3–7.4 and osmolarity ranging from 320–330 mOsm. Recordings were done at room temperature. The membrane potential was held at -60 mV throughout the recordings and with a voltage step of 10 mV was applied prior to NMDA application to monitor series resistance. Recordings were rejected if a series resistance change greater than 10% was observed. The intracellular solution consisted of 11 mM Ethyleneglycol-bis-(α-amino-ethyl ether) N,N'-tetra-acetic acid (EGTA) as intracellular calcium chelating buffer, 10 mM HEPES, 2 mM MgCl2, 2 mM Tetraethyl ammonium chloride (TEA-Cl) to block K+ channel, 1 mM CaCl2, 140 mM CsF, and 4 mM K2ATP. NMDA currents were evoked by rapid application of NMDA solution delivered from a multi-barreled fast perfusion system for 2 seconds in every minute. The solution was delivered at a rate of approximately 1 ml per minute. Abl or heat-inactivated Abl (0.5 μg/mL) was added to the ICF.
Immunoprecipitation and western blotting
The CA1 region of the hippocampus was microdissected from day 14–22 Wistar rat pups. CA1 slices (5–10 per condition) were incubated for 10 minutes with vehicle or PDGF-BB. Slices were washed in ice-cold ECF and homogenized in solubilization buffer (20 mM Tris, pH = 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 30 mM Na pyrophosphate, 1 mM βglycerophosphate, 1% Triton (1% NP-40 was substituted for immunoprecipitation), 1 mM Na3VO4, and MINI cocktail (Roche, Mannheim, Germany). Samples were centrifuged at 14,000 × g for 20 minutes and lysates were used for protein determination. Total protein concentration was determined by BCA Protein Assay (Pierce, Rockford, Il, USA). For Western blot, equal amounts of protein were loaded for each sample. For immunoprecipitations, lysate protein concentrations were normalized and lysates were incubated with primary antibody and protein A/G beads (Sigma) overnight at 4°C. Beads were washed three times and boiled in SDS loading buffer for 5 minutes before separation by SDS page electrophoresis. Proteins were transferred to nitrocellulose membranes, blocked with 5% non-fat dry milk in Tris-buffered saline for 1 hr at room temperature or overnight at 4°C, and incubated in primary antibodies for 1 hr or overnight at 4°C. Membranes were washed three times in Tris-buffered saline with 0.1% Tween-20, incubated with HRP-conjugated secondary antibodies for 1 hr, washed again, and bound antibodies were visualized by the enhanced chemiluminescence method. Densitometric analysis of Western blots was performed using the Kodak Image Station 2000R software.
Hippocampal tissue was incubated with vehicle or PDGF-BB and the tissue was dounce homogenized in fractionation buffer 1 to yield the S1 fraction (buffer 1: 0.32 M sucrose, 4 mM HEPES, pH 7.3, MINI protease inhibitor cocktail 1:10, and 1 mM Na3VO4). The homogenized tissue was centrifuged at 1000 × g for 10 minutes at 4 degrees. The supernatant was retained and centrifuged at 14,000 × g for 20 minutes. The supernatant (S2) was retained and the insoluble material was resuspended in fractionation buffer 2 (buffer 1 + 1% triton). The resuspended mixture was centrifuged at 100,000 × g for 60 minutes and the supernatant (S3, triton-soluble membrane fraction) was retained. The pellet (P3) was resuspended and dissolved in fractionation buffer 3 (buffer 1 + 0.5% SDS + 1% deoxycholate). Protein concentration was determined by BCA protein assay and lysates were subjected to Western blotting as described above.
Statistical analysis of the data was completed using Prism® GraphPad program. Graphs and sample tracings were made from Origin® program. All data was reported as mean ± SEM. Significance level was set at α = 0.05. Data was analyzed by Student's t-test or unpaired Student's t-test where appropriate.
All animal experiments were performed in agreement with the guidelines of the policies on the Use of Animal at the University of Toronto.