Anti glyceraldehyde 3 phosphate dehydrogenase gapdh

Met, CQ, TTC, Evans Blue, dimethyl sulfoxide (DMSO), TMRE, MDC, PI, Hoechst was obtained from Sigma-Aldrich (St. Louis, MO, USA). Miransertib was purchased from Selleck Chemicals (Houston, Texas, USA).DMEM and FBS were purchased from Gibco Laboratories (Life Technologies, Inc., Burlington, ON, Canada).CCK-8 was purchased from Dojindo Laboratories (Tokyo, Japan).The commercial assay kits for the CK-MB were from Jiancheng Bioengineering (Nanjing, Jiangsu, China). The primary antibodies used were anti-IL-1β, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Bioworld (MN, USA), anti-IL-6 (Abcam, Cambridge, MA, USA), anti-Bax, anti-phospho-Akt, anti-Akt, anti-phospho-mTOR and mTOR, anti-phospho-AMPK and AMPK, anti-Beclin-1, anti-P62, anti-Atg5 were purchased from Cell Signaling Technology (Beverly, MA, USA). In addition, anti-LC3B from Sigma and anti-Bcl-2 from Absin were used. All the antibodies were from rabbits and dilutions of antibodies were 1:1000, except anti-GAPDH (1:5000).

The mouse brains were rapidly dissected on ice, and the hippocampal and prefrontal cortices were removed for western blotting. The tissues were homogenized using T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, United States) plus Pierce Protease and Phosphatase Inhibitor Mini Tablets (Thermo Fisher Scientific, Waltham, MA, United States). The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis, transferred onto polyvinylidene difluoride (PVDF) membranes, and incubated overnight in primary antibodies. Except for the anti-tau 5 (ab80579; Abcam, Cambridge, United Kingdom) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Bioworld, St. Louis, Park, MN, United States) antibodies, the rest were the same as those in immunofluorescence experiments. The bands were analyzed using the ImageJ software.

The cells were harvested and lysed in RIPA lysis buffer (Thermo Scientific, Rockford, IL, USA) supplemented with a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Protein from each sample was resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk (Bio-Rad) in Tris-buffered saline containing 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with anti-pAKT (1:1000, Cell Signaling Technology, Inc., Beverly, MA, USA), anti-AKT (1:1000, Cell Signaling Technology), anti-Bax (1:1000, Cell Signaling Technology), anti-Cleaved caspase 3 (1:1000, Cell Signaling Technology), anti-Bcl-2 (1:1000, Abcam, Cambridge, UK), anti-glyceraldehyde-3 phosphate dehydrogenase (GAPDH) (1:5000; Bioworld, Nanjing, China) primary antibody at 4 °C overnight. After washing with TBST, the membranes were incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Following three additional washes with TBST, the protein bands of interest were visualized using enhanced chemiluminescence detection reagents (Thermo) and the Bio-Rad Gel Doc/ChemiDoc Imaging System and then analyzed using Quantity One software.