Ab18258

Immunolabeling of NPCs and derived neurons was accomplished by utilization of a standard two-day protocol with: 15 min fixation in 4% PFA, 10 min permeabilization in 0.1% triton-PBS and 30 min blocking with 5% donkey serum. Following dilutions of antihuman primary antibodies in PBS were applied overnight at 4 °C: Nestin (Millipore, MAB5326, 1:200), SOX2 (Abcam, ab97959, 1:200), Doublecortin (Cell Signaling, 4604S, 1:200), MAP2 (Millipore, MAB3418, 1:200), PSA-NCAM (Millipore, MAB5324, 1:200), PSD95 (Abcam, ab18258, 1:200) Tubulin, beta 3 (Millipore, MAB1637, 1:200). For fluorescence staining, we incubated samples for 2 h with Alexa-488 and Alexa-594 conjugated donkey antibodies (1:200) against mouse and rabbit immunoglobulins (Supplementary Figs 6 and 7 are representative images from two sets of immunolabelled cultures of NPCs and neurons).

Protein lysates from cortical neurons, 3xTg-AD mice and human brains were separated by SDS-PAGE using 7.5% Tris-Glycine polyacrylamide gels (Bio-Rad). Electrophoresis was conducted in a Tris-Glycine buffer (25 mM Tris, pH 8.3, 192 mM glycine, 0.1% SDS in dH₂O) by using the Criterion cell system (Bio-Rad). Blots were developed with rabbit anti-NR2B (1:1000, Millipore, #AB1557P), rabbit anti-pSer¹³⁰³NR2B (1:1000, Millipore, #07-398), rabbit anti-NR2A (1:1000, Millipore, #AB1555P), rabbit anti-PSD95 (1:500, abcam, #ab18258), mouse anti-synaptophysin (1:500, Millipore, #MAB329), rabbit antiphosho PKC (1:1000, Cell Signaling, #9371), rabbit anti-PKC (1:1000, abcam, #ab179521) and rabbit anti-β-actin (1:5000; Sigma-Aldrich, #A2066). Secondary antibodies conjugated with horseradish peroxidase (HRP) were purchased from Sigma (1:5000, sheep antimouse-HRP #A6782, goat antirabbit-#HRP A6154).

Proteins of primary cultured neurons and homogenized hippocampal tissues were extracted in RIPA buffer (ThermoFisher) supplemented with a protease and phosphatase inhibitor cocktail (ThermoFisher). After centrifugation (4 °C, 13,000 rpm, 15 min), the supernatant was collected into a new 1.5 mL tube and stored at − 80 °C. Total proteins (15–30 μg) were resolved on 10–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and electrotransferred with transfer buffer to polyvinylidene fluoride transfer membrane (Bio-Rad). Then, the membranes were blocked in TBST (10 mM Tris- HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) containing 5% BSA and incubated with the appropriate primary and secondary antibodies. PSD-95, MAP2, GFAP, Iba1 and Nlgn3 were detected in the membranes using anti-PSD95 (Abcam, ab18258), anti-MAP2 (Sigma-Aldrich, M9942), anti-GFAP (Millipore, MAB360), anti-Iba1 (Invitrogen, PA5-27436), and anti-Nlgn3 (Santa Cruz, SC-271880), respectively. β-Actin antibody (Sigma-Aldrich, A1978) was used as the standard. Protein bands were detected using the Clarity™ Western ECL Substrate (Bio-Rad). Bands were captured using Image Lab 6.0.1 (Bio-Rad). Densitometry analyses are presented as the ratio of protein to β-actin protein, which was compared with the controls and normalized.