Ab181869

Mouse tissues were homogenized and epididymal sperm were sonicated in RIPA buffer with protease inhibitor (A32963, Pierce) and 1 mM PMSF. Tissue protein lysates were separated by 4–20% SDS-PAGE gel and transferred to polyvinlylidenedifluoride (PVDF) membranes (Bio-Rad). The membranes were blocked in 5% non-fat milk, subsequently incubated with primary antibodies in blocking solution overnight at 4 °C. After washed in TBST, the membranes were incubated with HRP conjugated goat anti-rabbit IgG (1:5000, 1706515, Bio-Rad) or HRP conjugated rabbit anti-goat IgG (1:5000, 1721034, Bio-Rad) for 1 h. Membranes were incubated with chemiluminescent substrates (Bio-Rad) for detection in a molecular Imager ChemiDoc XRS+ imaging system (Bio-Rad) after 3 washes in TBST. β-Actin was visualized by incubating with anti-β-Actin-HRP (1:10000, A3854, Sigma-Aldrich) after stripping and washing. The primary antibodies used are as follows: rabbit anti-SYPL1 (1:2000, Abcam, ab184176), rabbit anti-VAMP2 (1:10000, ab181869, Abcam), rabbit anti-VAMP3 (1:10000, ab43080, Abcam), rabbit anti-VAMP4 (1:5000, 10738-1-AP, Proteintech), rabbit anti-AIF (1:1000, 5318, Cell Signaling Technology), rabbit anti-HK1 (1:4000, ab3543, Millipore), rabbit anti-PGK2 (1:5000, ab183031, Abcam), goat anti-PRSS21 (1:500, PA5-47879, ThermoFisher).

Animals were euthanized and perfused with 4% PFA in 0.1 M phosphate buffer, pH 7.4. Their brains were removed and post-fixed for 1 h, followed by cryoprotection in 30% sucrose in TBS containing 0.02% sodium azide. The harvested brain tissues were cryo-sectioned at a thickness of 16 μm along the sagittal or coronal plane. The sections were stained with the following primary antibodies: antibodies against VAMP2 (1:400, Abcam, ab181869, Cambridge, UK) and human pSer129 α-Syn (1:1000; FUJIFILM Wako Pure Chemical Corporation, 015-25191, Tokyo, Japan), overnight at 4 °C to detect the interactions between the VAMP2 and human pSer129 α-Syn proteins. After rinsing, the sections were simmered with the secondary oligonucleotide-linked antibodies (The Duolink® kit, DUO92102, Olink Bioscience, Uppsala, Sweden) provided in the kit. The oligonucleotides attached to the antibodies were detected using a fluorescent probe (Detection Kit 563). The specks were detected using confocal imaging (LSM700, Zeiss, Oberkochen, Germany). The samples were analyzed and figures prepared using ZEN software (ZEN 3.0 blue edition, Zeiss, Oberkochen, Germany).

Antibodies against GluR1 (ab31232), GluR2 (ab133477), GFP (ab1213, ab183734), PSD95 (ab2723, ab13552), synapsin-I (ab64581), syntaxin-2 (ab233275), synaptotagmin (ab13259), VAMP2 (ab181869), neuroligin-1 (ab153821), α-tubulin (ab7291), and MAP2 (ab5392) were purchased from Abcam. Secondary antibodies used for immunocytochemistry were: Alexa 488-labeled chicken anti-mouse or anti-rabbit; Alexa 594-labeleddonkey anti-mouse or anti-rabbit (Invitrogen, Eugene, OR); all used at a dilution of 1:500. 4′, 6-Diamidino-2-phenylindole (DAPI) (4′, 6′-diamidino-2-phenylindol) (Invitrogen) was used as a nuclear counterstain at 1 µg/ml. CTZ was purchased from Tocris Bioscience, and Furo-2, AM (F1201) was ordered from ThermoFisher. Bicuculline (S2694) and 5-Fluoro-2′-deoxyuridine (FDU) (F3503) were purchased from Sigma Aldrich.