Ab5666

Protein extraction from hippocampi and immunoblots of UPR and synaptic proteins were performed as described [23 (link), 24 (link)]. Proteins were detected with the following antibodies: eIF2α-P (S51) (1:1000; 9721, Cell Signaling) and eIF2α (L57A5) (1:1000; 2103, Cell Signaling), ATF4 (CREB-2, 1:1000; sc-200, Santa Cruz), GADD34 (1:1000, 10449-1-AP, Proteintech), PERK-P (1:200, 32577, Santa Cruz), PERK (1:1000, 3192, Cell Signaling), SNAP25 (1:10,000, ab5666, Abcam), PSD-95 (1:1000, EP2652Y, Millipore) total tau (Tau 5, 1:5000, AHB0041, Invitrogen), GSK3β (1:2000, 9832, Cell Signaling), pSer⁹-GSK3β (1:1000, 9322, Cell Signaling) and pTyr^279/216-GSK3α/β (1:1000, 05-413, Millipore). Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000; DAKO) were applied, and protein was visualised using enhanced chemiluminescence (GE Healthcare) and quantitated using ImageJ. Antibodies against GAPDH (1:5000; sc-32233, Santa Cruz) and Beta-III-tubulin (1:5000, MAB1637, Millipore) were used to determine loading.

For Western blot, the procedures of electrophoresis, transfer, and immunodetection were performed according to our previous study (Xu et al., 2012 (link); Hu et al., 2013 (link)). The primary antibodies used were as follows: antibody for the Ras-related protein Rab-3A (Rab3a, ab3335, 1:2000); syntaxin-binding protein 1 (Stxbp1, Munc18-1, ab124920, 1:4000); synapsin-1 (Syn1, ab18814, 1:1000); syntaxin-1A (Stx1a, ab41453, 1:3000); synaptosomal-associated protein 25 (SNAP25, ab5666, 1:4000); vesicle-associated membrane protein 2 (VAMP2, ab3347, 1:2000); synaptophysin (ab52636, 1:1000) (all purchased from Abcam); synaptotagmin 1 (Syt1, Millipore MAB5200, 1:1000); syntaxin-1B (Stx1b, Synaptic Systems 110402, 1:1000); and PSD95 (CST #3450, 1:1000). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG (purchased from Bio-Rad, dilution 1:15000) were used as secondary antibodies. After immunodetection, the intensity of the immunostained bands were normalized for the total protein intensities measured by Coomassie blue from the same blot (Van den Oever et al., 2008 (link); Counotte et al., 2010 (link)). The images were subjected to densitometric analysis performed using Quantity One software (Bio-Rad).