Jen et al. p1
NEUROLOGY 2005/101865
Supplemental Information
Functional Studies:
Site-directed mutagenesis. We obtained and transferred the full-length cDNA for wild type SLC1A3 (IMAGE:5264000) into a mammalian expression vector pcDNA3.1 (Invitrogen). We generated the mutant construct using QuickChange (Stratagene). Both the wild type and the mutant clones were fully sequenced for confirmation.
Cell culture and transfection. COS7 cells were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 8-10% fetal bovine serum and maintained at 37ºC with 5% CO2. EAAT2 and EAAT3 cDNA in pBS (generous gifts from S. Amara) were transferred to pcDNA3.1. EAAT2, EAAT3, wild type and mutant EAAT1 constructs in pcDNA3.1 (Invitrogen) were transfected with LipofectAmine 2000Plus (GIBCO). 1-2 days after transfection, cells were dissociated using enzyme-free cell dissociation buffer (GIBCO) and plated for glutamate uptake assay and immunocytochemistry.
Glutamate uptake assay. Cells attached to 60-mm-diameter culture dishes were incubated with 1.5ml of 1 mM L-glutamic acid containing 1 mCi/ml L-[3,4-3H]-glutamic acid at room temperature for 2 min. Glutamate uptake was linear with time during this period. The transporter function was assayed as previously described 16.
Protein extraction and Western blot analyses: Total protein was obtained by lysing the cells with a slightly modified RIPA buffer containing 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 0.2 mM Pefabloc SC, 0.1 mg/ml benzamidine and 8 µg/ml each of calpain I and II 17. Protein concentration was determined with the Micro BCA protein assay reagent kit (Pierce Chemical). Total protein (10 µg per lane) was subjected to SDS-polyacrylamide gel electrophoresis and transferred to NitroBind Pure Nitrocellulose membranes (Osmonics, Inc.). The membranes were exposed to monoclonal anti-EAAT1 antibody (Novocastra Laboratories) and monoclonal anti-actin antibody (Chemicon International) for 3 hrs in separate incubations. Incubation with the secondary antibody, horseradish peroxidase conjugated horse anti-mouse, followed for 1 h at room temperature. The incubation buffer was phosphate-buffered saline (PBS) with 5% nonfat dry milk and 0.1% Tween-20 detergent and the washes throughout the procedure were carried out with PBS plus 0.1% Tween-20. The stripping buffer was 62.5 mM Tris-HCl at pH 6.8, 2% SDS and 100 mM 2-mercaptoethanol, and stripping was performed between the EAAT1 and actin analyses for 30 min at 50˚C. The proteins were visualized with the Western Lightning Chemiluminescence Reagent Plus Kit (Perkin Elmer) on Hyperfilm autoradiography film (Amersham Life Science). Equivalent volumes were loaded for the membrane fractions before the protein was subjected to electrophoresis through a 4-20% gradient gel. The blots were probed with anti-actin antibodies as a loading control. For the blot, anti-EAAT1 antibody was used at a dilution of 1:200 and anti-actin antibody was used at 1:2000. For both analyses, the secondary antibody was used at 1:3000.
Biotinylation: Cell surface proteins were biotinylated and partially purified as previously described, with modifications 18. The cells were washed three times with ice-cold PBS, dissociated with 0.5 ml cell dissociation buffer per 6 cm culture plate, and transferred to microcentrifuge tubes. Low speed centrifugation followed for 3 min at room temperature and the supernatant was removed. The cells were resuspended in 1 ml PBS and 200 µl of 10 mM biotin (Sulfo-NHS-Biotin, Pierce Chemical) per ml cell suspension were added. The mixture was incubated with gentle rocking in the cold room for 30 min. After low speed centrifugation for 3 min at 4˚C, the biotin solution was removed and the cells were washed twice with ice-cold PBS containing 100 mM glycine. After the last wash, incubation followed in the same buffer for 30 min at 4˚C with gentle rocking to quench any unbound biotin. Another centrifugation round followed, and the supernatant was discarded. The cells in the pellet were lysed as specified above. A sample called “Total Cell Lysate Fraction” was removed at this point and the protein concentration was determined. An equal volume of ImmunoPure Immobilized Monomeric Avidin (Pierce Chemical) was added to the remaining mix and it was incubated with gentle mixing on a rocker for 1 hr at room temperature. The mix was centrifuged and the supernatant removed. The pellet was washed four times with 1 ml RIPA lysis buffer containing protease inhibitors. After the final wash, the pellet was resuspended in Laemmli buffer (0.025M Tris, 0.1% SDS, 0.192M Glycine), and the biotinylated proteins were eluted overnight at 4˚C. Centrifugation was then carried out for 10 min at 16,300 x g in the cold room. The supernatant was removed, and samples were stored at -20˚C. Western blot analysis was carried out as specified above.
Immunocytochemistry and confocal imaging. COS7 cells were co-transfected with EAAT1 and CD8 19. 1-2 days after transfection, the cells were fixed with 4% paraformaldehyde in PBS for 20 min and then blocked with 6% BSA for 30 min at 37ºC. Cells were incubated with a mouse anti-human EAAT1 antibody (Novocastra Lab. Ltd) and a rabbit anti-CD8 antibody (Abcam Inc.) for 1 hr at 37ºC, followed by incubation with Alexa 594-conjugated goat anti-mouse antibody (Molecular Probes, Inc.) and Alexa 488-conjugated goat anti-rabbit antibody (Molecular Probes, Inc.) for 1 hr at room temperature. Cells were mounted with Fluoromount-G (S. Biotech. Associates, Inc.). Epifluorescent microscopy was performed using Olympus BX-51. Confocal fluorescent microscopy was performed using a Leica TCS SP MP inverted confocal microscope at the UCLA Carol Moss Spivak Cell Imaging Facility. A 63X oil immersion objective was used for imaging. Data were acquired and analyzed with Leica Confocal Imaging Software and processed using Adobe Photoshop 6.0 (Adobe Systems Inc.).
E-Table 1. Results of quantitative rotational testing (0.05 Hz, peak velocity 60 deg/sec). VOR- vestibulo-ocular reflex; OKN- optokinetic nystagmus; VOR-Fix- fixation suppression of the VOR.
E-Figure 1. EEG of the patient obtained at baseline (January 2004), demonstrating bifrontal spike and slow wave discharges.