Snap25

Western blotting was performed on synaptosomes, as described previously [50 (link)]. Equal amounts of lysate protein were run on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred to polyvinylidene difluoride membrane (GE Healthcare UK Ltd., Amersham, UK). The membrane was first blocked with TBST buffer (500 mM NaCl, 20 mM Tris-HCl [pH7.4], and 0.1% Tween-20) containing 5% non-fat dry milk, and then incubated with primary antibody overnight at 4 °C and horseradish peroxidase-conjugated secondary antibody (1:2000, Gentex, Zeeland, MI, USA) for another 1 h. Bound antibodies were detected with an enhanced chemiluminescence (ECL) system. Quantification of the signals was obtained using Image software. From the obtained intensity for each band the background subtraction was made, and all the values were normalized to β-actin. The primary antibodies used were PKC (1:600, Abcam, Cambridge, UK); pPKC (1:1000; Cell Signaling, Beverly, MA, USA); PKCα (1:600; Cell Signaling, Beverly, MA, USA); pPKCα (1:2000; Abcam, Cambridge, UK); SNAP-25 (1:20,000; Abcam, Cambridge, UK); pSNAP-25 (Ser187) (1:2000; Abcam, Cambridge, UK); pMARCKS (Ser152/156) (1:250; Cell Signaling, Beverly, MA, USA); and β-actin (1:1000; Cell Signaling, Beverly, MA, USA).

The indicated amounts of recombinant GST(Sigma-Aldrich), SNAP25(abcam), SNAP23(Origene), SNAP29(Origene), VAMP3(LSbiosciences), VAMP3(MyBiosource), RALGDS(Origene), PIK3CG(Origene), or Raf1(Origene) were spotted on a nitrocellulose membrane and allowed to dry for 30 min. The membrane was then blocked in 5% milk in TBST and incubated overnight at 4 C with 1 μg KRAS protein (abcam). For RNA competition far westerns, in vitro transcribed RNA was preincubated with 1 μg KRAS protein at 4 C for 1 h prior to overnight incubation with the membrane. The membrane was then washed in 5% milk in TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at RT. The membrane was then developed using the SuperSignal West Dura reagent (ThermoFischer). Quantification was done using ImageJ. For loading controls, duplicate membranes were spotted and immediately stained with a Coomassie Stain Kit (Biorad).

Frozen hippocampal tissues were homogenized in ice-cold RIPA buffer (Sigma, USA) containing 1% protein inhibitor (Sigma, USA) and 1% phosphatase inhibitor (Sigma, USA). After centrifugation at 12,000 rpm at 4 °C for 15 min, the supernatant was collected for quantification by a BCA protein assay kit (Thermo Fisher Scientific) and then denatured in loading buffer. Equal amounts of protein (50 µg) were separated on 10% gels by SDS‒PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membrane was placed in 5% foetal bovine serum albumin (BSA) in washing buffer (Tris-buffered saline containing 0.1% v/v Tween-20), blocked at room temperature for 2 h, and then incubated overnight at 4 °C with primary antibodies [GAPDH (rabbit, 1:1000, Cell Signaling Technology, USA), β-actin (rabbit, 1:1000, Santa Cruz, USA), BDNF (1:1000, Abcam, UK), Nrf2 (rabbit, 1:1000, Proteintech, USA), DMT1 (rabbit, 1:1000, Abcam, UK), TfR (rabbit, 1:1000, Abcam, UK), Tf (rabbit, 1:1000, Abcam, UK), PSD95 (mouse, 1:200, Abcam, UK), and SNAP25 (rabbit, 1:1000, Abcam, UK)] followed by incubation with horseradish peroxidase-linked secondary antibodies for 2 h at 4 °C. Chemiluminescence detection was measured using ECL reagents (Millipore, Billerica, MA, USA). The bands were scanned, and their densities were analysed by ImageJ.

All chemical reagents and solutions were purchased from Fisher Scientific (Gll chem, Sweden), Saveen & Werner (Limhamn, Sweden) or Sigma-Aldrich (Stockholm, Sweden) unless otherwise stated. Commercially available primary antibodies against CD3 (Bio-techne, UK), PSD-95, SNAP-25, NeuN and BDNF (Abcam, UK), CD68 (Invitrogen, Sweden), and Iba-1 (Wako, Japan) were used for immunofluorescence. Secondary antibodies Alexa Fluor 488 donkey anti-mouse, anti-rabbit, goat anti-rabbit, or goat anti-rat Fluor 594 (Nordic Biosite, Sweden) were used for visualization. Primers for qPCR were purchased from Eurofins (Ebersberg, Germany).

Neurons were fixed at DIV18 with 3.7% formaldehyde (Merck) in phosphate-buffered saline ([PBS] 137mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 [pH 7.4]) for 20 minutes. Cells were permeabilized in 0.5% TritonX-100 (Fisher Chemical) for 5 minutes and blocked with 0.1% TritonX-100 and 2% normal goat serum for 60 minutes. Primary antibody incubation with MAP2 (Abcam, 1:1,000), SMI312 (Biolegend, 1:1,000), CaMK2 (SantaCruz, 1:350), VGLUT1 (Millipore, 1:5,000), BDNF (DSHB, 1:10) proBDNF(Alomene Labs, 1:300), CHGB (SySy, 1:500), SCG2 (Biodesign International, 1:1,000), synaptophysin 1 (SySy; 1:1,000), VAMP2 (SySy, 1:1,000), synaptotagmin 1 (W855 a kind gift from T. Südhof, Stanford, CA; 1:2000), MUNC18-1 (BD, 1:1,000), SNAP-25 (Abcam, 1:1,000), phosphoCREB-S133 (Abcam, 1:200) was performed for 2 hours at RT. Alexa Fluor conjugated secondary antibodies (1:1,000; Invitrogen) were incubated for 1 hour at RT. Coverslips were mounted in Mowiol and imaged on Nikon Eclipse Ti microscope confocal laser-scanning microscope (40× objective; NA 1.3) with NisElements 4.30 software. Images were acquired as Z-stack of 3 planes 250 μm apart and 4 images per plane with 15% overlap to ensure that the entire micro-island was in the field of view.

The following primary antibodies were used in this study: Estradiol (BioGenex, San Francisco, CA, USA #AR038), NeuN (Millipore Biotechnology, Massachusetts American #NG1857584), DCX (Santa, TX, USA #sc8066), SOX2 (Abcam, Cambridge, UK #ab97959), GFAP (Abcam, #ab53554), Iba1 (Abcam, #ab175076), MAP2 (Santa, #sc8066), MBP2 (Proteintech, Chicago, IL, #NO.10458-1-AP), GAPDH (Proteintech, #NO.60004-1-1g), p-CREB (Cell-signaling, Boston, MA, USA #9198s), CREB (Abclonal, Wuhan, China #56653), BDNF (Arigo, Shanghai, China #56653), PSD95 (Abcam, #ab1825), Neurofilament (Abcam, #ab7794), Spinophilin (Abcam, #ab13359) and SNAP25 (Abcam, #ab5666).

After perfusion with 4% paraformaldehyde, the brains were removed and fixed with 4% paraformaldehyde overnight. Next, they were dehydrated in an ethanol gradient (50%, 70%, 80%, 90%, 95%, 100%) and xylene. The tissues were embedded in paraffin wax and sliced into 4-μm sections that were mounted on slides. The slides were deparaffinized and rehydrated in xylene and an ethanol gradient. Antigens were retrieved via thermal repair in a citrate buffer. Then, the slides were blocked at room temperature for 30 min and incubated with the primary antibody of GAP43 (1:500, Abcam) or SNAP25 (1:500, Abcam) overnight at 4 °C. After washing, the slides were incubated with secondary anti-rabbit IgG H&L (Cy3) antibody (preabsorbed and used at 1:500, Abcam) for 1 h at room temperature. Finally, the slides were counterstained with DAPI (Beyotime, Shanghai, China) and sealed with anti-fluorescence quenching sealing tablets (Yeasen, Shanghai, China). Images were obtained using a confocal microscope (Leica TCS SP5 II).