RNA immunoprecipitation and real-time quantitative PCR STAU1 or STAU2 antibodies (MBL International, Woburn, MA) were conjugated to magnetic DynaBeads (Life Technologies) according to the manufacturer’s instructions. Differentiated SH-SY5Y cells were gently harvested by adding chilled lysis buffer containing protease and RNAse

inhibitors to the cultures for 5 min. The lysate was collected and an aliquot was saved for analysis of total RNA, while the remainder was incubated with antibody-conjugated DynaBeads for 1 h at 4°C. The beads were then washed and spiked with synthetic RNA as an internal control. RNA was simultaneously isolated from the immunoprecipitated samples and total lysate aliquots Inhibitors,research,lifescience,medical by Trizol-chloroform extraction. The concentration and purity of RNA were determined using an ND1000 spectrophotometer (NanoDrop Technologies, Wilmington, Inhibitors,research,lifescience,medical DE). Synthesis of cDNA was carried out using High Capacity cDNA see more Reverse Transcription Kit (Applied Biosystems, Foster City, CA), with 1 μg of RNA per 20-μL reaction. For real-time quantitative Inhibitors,research,lifescience,medical PCR (RT-qPCR), 100 ng of the cDNA was used in 5-μL reactions with PerfeCTa SYBR Green FastMix (Quanta Biosciences, Gaithersburg,

MD). Reactions were carried out in triplicate in a LightCycler 480 II thermal cycler (Roche, Indianapolis, IN). Cycling conditions followed the manufacturer’s suggestions in the SYBR Green kit instructions. All qPCR results were normalized to the spiked synthetic RNA or endogenous Inhibitors,research,lifescience,medical hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA expression and analyzed using Absolute Quantification Software (Roche). Imaging Fluorescence imaging of primary cultures and stained sections were Inhibitors,research,lifescience,medical performed on a Zeiss LSM 5 Pascal laser confocal inverted microscope equipped Ar and HeNe lasers. AlexaFluor-488, -546, and -633 secondary antibodies with directly conjugated fluorophores were used to detect primary antibody signals. Images were acquired using

the included LSM software, and were analyzed using ImageJ. Bright-field imaging below was performed using an upright Zeiss AxioScope 2 Plus microscope equipped with an ASI motorized stage and Zeiss Axiocam MRc camera. Statistical analysis Data were analyzed using Microsoft Excel (Redmond, WA), and plotted using GraphPad Prism software (San Diego, CA). Unpaired t-tests were performed for protein expression in synaptosome experiments and mRNA expression in RT-qPCR experiments. The significance criteria were set at P < 0.05 for statistical measures. Results Selenoprotein W is abundant in the mammalian brain and its mRNA is found in mouse brain neurons; however, the cellular location of the protein has not been described. We sought to determine whether Sepw1 is expressed in neurons of mouse brain, and if neuronal expression of Sepw1 is reduced in Sepp1−/− mice.