Anti rabbit or anti mouse igg hrps

Hippocampal proteins were extracted from each group using lysis buffer (Beyotime, China) and phosphatase inhibitor cocktail (Thermo Fisher Science, USA). Protein samples were mixed with loading buffer, heated at 100° C for 5 min, then separated on 10% SDS-PAGE and transferred to PVDF membranes. Then, blots were blocked with 5% nonfat dried milk for 2 h at room temperature. Monoclonal anti-Ppp1c (diluted 1 : 2000, molecular weight: 39 kDa, Abcam, USA), monoclonal anti-Arsa (diluted 1 : 2000, molecular weight: 54 kDa, Abcam, USA), anti-Tau (phospho S262 + T263) (diluted 1 : 2000, molecular weight: 79 kDa, Abcam, USA), anti- total Tau (diluted 1 : 2000, molecular weight: 52 kDa, Abcam, USA) or monoclonal anti-β-actin (diluted 1 : 1000, molecular weight: 42 kDa, Santa Cruz, USA) were used as primary antibodies. Anti-rabbit or anti-mouse IgG HRPs (diluted 1: 3000, Thermo Fisher Scientific, USA) were used as secondary antibodies. After washing in TBST, the membranes were exposed to chemiluminescence reagents using an electronic chemiluminescence kit on a phosphor imager (Pierce Biotechnology, USA) and analyzed for optical density based on Java (ImageJ) software (National Institutes of Health, Bethesda, MD, USA).

Each sample from the above groups was extracted with 400 ml RIPA lysis buffer (Beyotime, Haimen, Jiangsu, China) with protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA, USA). BCA protein assay kit (Thermo Scientific, USA) was used to determine protein concentration. Equal amounts of proteins were separated by 10% SDS-PAGE gels and then transferred onto PVDF membranes. Membranes containing the transferred proteins were blocked for 1.5 h in 5% skim milk in TBST. Primary antibodies, anti-GRP78 (1:1000, Santa Cruz, sc-376768), anti-protein deglycase (DJ-1) (1:10,000, Abcam, ab76008), anti-dynamin-1 (1:1000, Abcam, ab52611), anti-14–3–3Z (1:1000, Abcam, ab155037), anti-NDUFV2 (1:10,000, Abcam, ab183715), anti-PERK (1:1000, CST, 3192S), anti-p-PERK (1:1000, CST, 3179S), anti-eIF2α (1:1000, Santa Cruz, sc-133132), anti-p-eIF2α (1:1000, CST, 3597S), and anti-CHOP (1:1000, CST, 2895S) were then added and incubated on ice overnight. After washing with TBST, membranes were incubated with anti-rabbit or anti-mouse IgG HRPs (Thermo Fisher Scientific, 1:3000) for 50 min at room temperature. Then the membranes were washed with TBST and treated with enhanced chemiluminescence (ECL) reagents from an ECL kit (Pierce, Thermo Scientific). Blots were detected on a phosphorimager and analyzed according to ImageQuant 1D software (GE Healthcare, USA).

The primary antibodies, anti‐ITPR1 (1:2000, Affinity, DF3000), anti‐ITPR2 (1:2000, Affinity, DF13336), anti‐FINC (1:2000, Abcam, ab45688), anti‐GP1BA (1:2000, Abcam, ab134087), anti‐ADAM10 (1:2000, Affinity, AF5294), anti‐CD63 (1:2000, Abcam, ab134045), anti‐PHB (1:3000, Abcam, ab75766), anti‐RAP1A (1:2000, Affinity, DF6157), anti‐UQCRH (1:1000, Abcam, ab154803) were added and incubated on ice overnight. After washing with TBST, the membranes were incubated with anti‐rabbit or anti‐mouse IgG HRPs (Thermo Fisher Scientific, 1:3000) for 50 min at room temperature. Then the membranes were treated with enhanced chemiluminescence (ECL) reagents from an ECL kit (Pierce, Thermo Scientific) for exposure.