Brainstem tissues were maintained for 1 hr at RT in aCSF, bubbled with 95% O2 and 5% CO2, containing Rp-cGMPS (3 μM, dissolved in 0.1% DMSO), PTIO (100 μM, with 0.1% DMSO), or none of them (control, with 0.1% DMSO). After measuring wet weight of individual brainstem click here tissue samples, they were homogenized in 0.32 M sucrose, 4 mM HEPES-NaOH (pH 7.3 kept at <4°C), supplemented with EDTA-free protease inhibitor cocktail (Roche), phosphatase Inhibitor Cocktail 3 (Sigma), and kinase inhibitor sodium orthovanadate

(Sigma). For lipid extraction, an ice-cold methanol/chloroform (2/1) mixture was added to each pellet. Samples were centrifuged at 1,500 rpm for 5 min, and then supernatants were removed to extract neutral lipids. To extract acidic lipids, an ice-cold methanol/chloroform/HCl 12N (80/40/1) mixture was added to the remaining tissue pellets, vortexed thoroughly, find more and centrifuged at 1,500 rpm for 5 min. Supernatants were collected and a chloroform/0.1N-HCl (1/2) nonpolar/polar solvents mixture was added to create a phase split of liquid to separate lipids from remaining other constituents. The chloroform phase (lower phase) comprising lipids was collected and evaporated by a speed-vacuum centrifuge (Eppendorf Concentrator

5301). Dried lipids samples were then rapidly dissolved again in PBS containing the PIP2 sensor provided in the PIP2 Mass ELISA kit (K-4500, Echelon). PIP2 in each sample was then quantified following the manufacturer’s protocol. Luminometric analyses were performed by measuring the final signal absorbance at 450 nm using a microplate reader (Benchmark Plus 170-6930J1, Endonuclease Bio-Rad). PIP2 levels were normalized to the wet weight of brainstem tissue. Brainstem, heart, and liver tissues of P7 and P14 rats were homogenized in ice-cold buffer containing 50 mM Tris, 1 mM EDTA, 150 mM NaCl, 1% NP-40, and a protease inhibitor cocktail (Roche). Homogenates were then centrifuged at 12,000 rpm for 30 min. The protein concentrations of lysates were measured using Micro BCA Protein assay kit (Thermo Scientific) and equal amount of proteins were loaded onto a SDS polyacrylamide gel. After separation by

electrophoresis proteins were transferred to a PVDF membrane (Bio-Rad). Blotted membranes were blocked with 5% nonfat dry milk in Tris-buffer saline with 0.1% Tween-20. Proteins were detected with the specific primary rabbit antibodies anti-PGKI (Abcam) and anti-alpha-tubulin (Sigma) prior to incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (Millipore). Protein immunoreactivity was further detected using ECL plus western blotting detection reagents (Amersham). Quantitative analyses were performed with a cooled CCD camera coupled with AE- 6971 Light Capture instrument (ATTO) and analyzed with the supplied CS-Analyzer software (ATTO). Data were analyzed using IGOR Pro 4 (WaveMatrics), MS Excel 2003 (Microsoft), and Sigmaplot 11 (Synstat Software) softwares.