Ecl kit

Cells were lyzed with a lysis buffer containing phenylmethyl sulfonylfluoride (PMSF) (Beyotime, Shanghai, China) at 4°C. Proteins were quantified using a BCA protein assay kit (ComWin Biotech, Beijing, China). Protein lysates (50 μg) were separated by 10% SDS-PAGE gels (Invitrogen) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Beyotime, Shanghai, China). The membranes were blocked with 5% non-fat dry milk in Tris-phosphate buffer containing 0.05% Tween 20 (TBS-T) for 1 h at room temperature and then treated with the primary antibodies at 4°C overnight. After washing with TBST, the membranes were incubated with peroxidase-conjugated affinipure goat anti-rabbit IgG (H+L) (ZSGB-BIO, 1:5000, ZB-2301) and peroxidase-conjugated affinipure goat anti-mouse IgG (H + L) (ZSGB-BIO, 1:5000, ZB-2305) for 1 h at room temperature. The blots were visualized using an ECL kit (ComWin Biotech, Beijing, China), and quantified using the Image J Software, normalized to β-actin. The primary antibodies were: E-cadherin (1:500, Cell signaling, #3195), Vimentin (1:1000, Cell signaling, #5741), ERK1/2 (1:1000, Cell signaling; #4695), p-ERK1/2 (Thr202/Tyr204) (1:1000, Cell signaling, #9101), α-catenin (1:500, Proteintech, Catalog number: 66221-1-Ig), β-catenin (1:1000, Santa Cruz, sc-7963), and β-actin (1:1000, Bioss).

Compound M1 were purchased from SPECS database. Anti-Bcl-2, anti-Cyt c, anti-COX-IV, anti-PARP, and anti-cleaved caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin was purchased from Proteintech. Mitochondria Isolation Kit for Cultured Cells (CAT. NO. 89874) was purchased from Thermo. JC-1 Assay Kit (CAT. NO. C2006) and Reactive Oxygen Species Assay Kit (CAT. NO. S0033) were purchased from Beyotime. The enhanced chemiluminescence (ECL) kit was purchased from Beijing Com Win Biotech Co, Ltd (Cwbio, China). CCK-8 reagent was purchased from Bimake (shanghai, China).

BV-2 microglial cells were lysed with radioimmunoprecipitation assay buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease inhibitors (Roche, Basel, Switzerland); 30 μg samples of the lysates were separated on 12% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies overnight at 4°C. The primary antibody incubation was followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The bound antibodies were detected using an ECL kit (ComWin Biotech Co., Beijing, China). Primary antibodies were as follows: p-p65 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:1000), p-IкBα (Abcam; 1:500), TLR4 (Santa Cruz; 1:1000), myeloid differentiation primary response 88 (MyD88; Santa Cruz; 1:1000) and β-actin (Santa Cruz; 1:1000). β-actin was used as a loading control.