The lysates were immunoprecipitated with Smurf1-specific antibodies and immunoblotted for the phosphorylation
level with anti-phosphor-(Ser/Thr) PKA substrate antibodies (Cell Signaling, Danvers, MA). For immunostaining, cultured hippocampal neurons were fixed with 4% paraformaldehyde for 12 min and then permeabilized in 0.3% Triton X-100 for 20 min and blocked with 1% BSA for 1hr. The fixed cells were processed further for immunostaining according to standard procedure and imaged with a confocal microscope (Leica DM IRBE) equipped with a 40× oil-immersion objective (NA1.0). Images were analyzed and processed for presentation in the figures, using brightness and contrast adjustments with NIH ImageJ software and following the guideline of Rossner and Yamada (2004). GW-572016 supplier Microfabrication and substrate coating methods followed those previously described (Hsu et al., 2005). Briefly, the poly(dimethylsiloxane) (PDMS) cuboids that were used to generate microchannels were prepared from Sylgard 184 base Selleckchem Raf inhibitor and curing agent (Dow Corning, Midland, MI). It was polymerized on a silicon wafer that is etched for patterns of parallel stripes (50 μm width each) spaced with 50 μm gaps. Solution containing the substrate factors was filled into the microchannels
formed by placing the PDMS cuboids over the poly-L-lysine-coated glass coverslip and overnight incubation allowed the substrate factor to be coated onto the coverslip. The substrate solutions were prepared with the following concentrations of the factors: fluorescently conjugated cAMP analog (Alexa Fluor 647 8-(6-aminohexyl) aminoadenosine 3″,5″-cyclicmonophosphate, tetra [triethylammonium] salt; F-cAMP), 20 μM; and BDNF, 0.5 ng/ml. In all coating solutions, 5 μg/ml of fluorescently-conjugated BSA was added as the marker for the found stripes. The method of in utero electroporation follows previously
described procedures (Saito and Nakatsuji, 2001), with minor modifications. Timed-pregnant Sprague-Dawley rats were anesthetized at E18 with isoflurane, and the uterine horns were exposed by way of a laparotomy. Saline solution containing the expression plasmid of interest (2 mg/ml) together with the dye Fast Green (0.3 mg/ml; Sigma) was injected (1–2 μl) through the uterine wall into one of the lateral ventricles of the embryos, and the embryo’s head was electroporated by tweezer-type circular electrodes across the uterus wall, and five electrical pulses (50 V, 50-ms duration at 100-ms intervals) were delivered with a square-wave electroporation generator (model ECM 830, BTX, Inc.). The uterine horns were then returned into the abdominal cavity, the wall and skin were sutured, and the embryos continued their normal development.