Gb113375

Western blotting was performed as previously described [26 (link)]. Proteins from the brain samples and cultured HT22 cells were lysed with RIPA lysis buffer. Equal amounts of protein (50 μg) were loaded onto a sodium dodecyl sulfate–polyacrylamide gel. The proteins were electrophoresed until they were fully separated and then transferred to a polyvinylidene difluoride membrane. The membrane was then blocked with 5% bovine serum albumin for 1 h at room temperature. The membranes were incubated with the following primary antibodies overnight at 4 ℃: anti-CCR2 (#12199, 1:1000, CST), anti-p-PI3K (#4228, 1:1000, CST), anti-PI3K (#AF7742, 1:1000, Beyotime), anti-AKT (#4691, 1:1000, CST), anti-pAKT (#4060, 1:1000, CST), anti-IL-1β (#ab254360, 1:1000, Abcam), anti-TNF-α (#ab255275, 1:1000, Abcam), anti-BAX (#GB114122, 1:1000, ServiceBio), anti-Bcl-2 (#GB113375, 1:1000, ServiceBio), anti-cleaved caspase 3 (#ab2302, CC3, 1:1000, Abcam), and anti-β-tubulin (#GB11017, 1:1000, ServiceBio). Suitable secondary antibodies (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA) were selected and incubated with the membrane at room temperature for 1 h. The bands were then observed using an enhanced chemiluminescence reagent. Image J software (NIH, Bethesda, MD, USA) was used for density measurements and quantification.

Paraffin-embedded xenograft sections were used for TDP-43 (Servicebio, China, GB112160, dilution: 1:400), ABHD2 (absin, abs140798, dilution: 1:200), C-caspase3 (Servicebio, GB11532, dilution: 1:500), Bcl-2 (Servicebio, GB113375, dilution: 1:500), and p53 (Servicebio, GB111740, dilution: 1:500) staining analysis. The antibody of Ki67 used in this experiment was purchased from Abcam (ab15580, dilution: 1:200). After deparaffinized and rehydrated, sections were laid in EDTA solution (pH9.0) for antigen retrieval by boiling in microwave oven for 8 min on medium heat. The slides were naturally cooled before treating with 3% H₂O₂ for 10 min. Sections were blocked in 10% normal goat serum for 30 min at room temperature, and then incubated overnight with indicated primary antibody at 4°C. After three washes with PBS, the slides were hybridized with the secondary antibody (Servicebio, GB23303, dilution: 1:200) for 50 min at room temperature. Finally, diaminobenzidine (DAB) (servicebio) was used for color development. After counterstaining with hematoxylin, dehydrate the slides and seal with neutral gum (Solarbio, China). Image J software was used for staining analysis.