Ab15458

The following primary antibodies were used: anti-GLT-1 raised against the peptide within 30 C-terminal amino acids from the canonical GLT-1 sequence (AB1783, EMD Millipore, Temecula, CA), and anti-GLT-1 raised against the peptide corresponding to residues 550 to the C-terminus of rat GLT-1 (ab41621, Abcam, Cambridge, MA), both recognizing GLT-1a. The GLT-1 antibody from Abcam was used in the co-IP experiments and the antibody from EMD Millipore for the immunofluorescent staining. Anti-GLAST (MABN794, Millipore, Temecula, CA); anti-EAAT3 (MAB1587, Millipore, Temecula, CA); anti-Na⁺/K⁺-ATPase (05-369, Millipore, Temecula, CA); anti-PS1 loop raised against the loop domain between transmembrane domains 6 and 7 of PS1, clone APS18 (ab15458, Abcam, Cambridge, MA) and anti-PS1 CT raised against the C-terminus of PS1 (mAb5643, Cell Signaling Technology, Danvers, MA and P7854, Sigma-Aldrich, St. Louis, MO). Alexa Fluor 488 (ThermoScientific, Waltham, MA) and Cy3-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were applied for the microscopy imaging and IRDye680/800- (Li-COR, Lincoln, NE) labeled ones were used for western blotting.

The samples were mixed with 2×SDS lysis buffer (50 mM Tris–HCl at pH 6.8, 2% SDS and 10% glycerol) with 1×Protein Inhibitor Cocktail, boiled at 100 °C for 10 min. The supernatant was obtained by centrifugation at 13,000 g for 10 min and the protein concentration was determined using the Pierce^TM BCA protein Assay kit (Thermo Fisher). Proteins in lysates were separated by SDS-PAGE, transferred to PVDF membranes, and blocked in 5% skim milk that contained 0.1% Tween 20 at room temperature for 1 h. The membranes were immunoblotted with the corresponding antibodies overnight at 4 °C, and then were washed and incubated with horseradish peroxidase conjugate secondary antibodies at room temperature for 1 h. After washing, the bands were visualized using the Pierce™ ECL Western Blotting Substrate kit (Thermo Fisher) and band intensities were quantified by ImageJ. The antibodies were listed as following: CNTNAP2 (ab33994, Abcam), Necdin (ab18554, Abcam), Flag-tag (14793, CST), HA-tag (3724, CST), GFP (2555, CST), CASK (ab252540, Abcam), GAPDH (97166, CST), Histone H3(9715, CST), PS1 (ab15458, Abcam).