Xrs imaging system

RIPA buffer (NCM biotech, China) added with 1% Protease Inhibitor Cocktail (NCM biotech, China) was used to lyse cells. BCA Protein Assay Kit (NCM biotech, China) was employed to determine protein concentrations. After transferred onto PVDF membranes, cell proteins were incubated with primary antibodies and specific HRP‐conjugated secondary antibodies successively. Signals were captured by XRS imaging system (Bio‐Rad, USA). Primary antibodies include: anti‐BCAT2 antibody (Cat: ab95976, Abcam, USA) and anti‐GAPDH antibody (Cat: ab8245, Abcam, USA). Secondary antibodies include: HRP goat anti‐rabbit IgG (Cat: 7074, Cell Signaling Technology, USA) and HRP goat anti‐mouse IgG (Cat: 7076, Cell Signaling Technology, USA).

Small intestinal tissue samples (approximately 20 mg) were lysed in an ice-cold RIPA lysis buffer, enriched with protease inhibitors. Subsequent protein concentration measurement was performed using a BCA protein assay kit. Proteins (20 µg per sample) were loaded into 10% polyacrylamide gels, separated through electrophoresis, and then transferred to nitrocellulose membranes. The protein-bound nitrocellulose membranes were blocked with a solution of 5% skim milk in PBS-Tween-20 (0.1%) for two hours at room temperature to avoid non-specific binding. The membranes were incubated with primary antibodies targeting p65 (1:1,000), p-p65 (1:1,000), TLR4 (1:1,000), Bcl-xl (1:1,000), cleaved caspase-3 (1:1,000), ZO-1 (1:1,000), Occludin (1:1,000), Claudin1 (1:1,000), E-cadherin (1:1,000) and GAPDH (1:4,000) at 4 °C overnight. This was followed by an hour-long incubation at room temperature with an HRP-conjugated secondary antibody. An ECL kit was employed to detect specific protein signals. Images of protein bands were captured with a Bio-Rad XRS imaging system and the intensity of the proteins was evaluated semi-quantitatively using ImageJ software.

Around 100 mg of mouse hippocampus tissue or the cultured HT22 and BV2 cells were lysed in the ice-cold RIPA lysis buffer supplemented with protease inhibitors, and the total protein concentrations were detected using BCA protein assay kit. The proteins (20 µg) from each sample were added to 10% polyacrylamide gels and separated and electrophoretically transferred to a nitrocellulose membrane. Non-specific binding in protein-transferred nitrocellulose membranes was blocked with 5% skim milk in PBS-Tween-20 (0.1%) for 2 h at room temperature. The membranes were incubated with anti-APP (1:1,000), BACE1 (1:1000), NF-κBp65 (1:1,000), p-NF-κBp65 (1:1000), Bax (1:500), Bcl-XL (1:500), caspase 3 (1:500), synaptotagmin 1 (1:1000), Hes1 (1:500), Notch (1:500) and GAPDH (1:2,000) antibodies, respectively, at 4 °C overnight, and then with HRP-conjugated secondary antibody for 1 h at room temperature. The specific protein signals were captured by ECL kit. The protein bands in images were visualized by Bio-Rad XRS imaging system, and the protein intensity was semiquantitatively evaluated by an observer in a blinded manner using image J software [14 (link), 33 (link)].

RIPA with protease inhibitor and phosphatase inhibitor (Sigma-Aldrich) was used to lyse BCa cells. After 30 min on ice, cells were centrifuged at 4 °C and the supernatant was the protein solution. After a bath in boiling water for 10 min, the protein solution was ready to test. Protein was separated in SDS-PAGE gels, then transferred to PVDF membrane (Millipore), blocked with 5% milk, and incubated with primary (Additional file 1: Table S1) and secondary antibodies (Additional file 1: Table S2) sequentially. Bands were developed with chemiluminescence kit (Bio-Rad) and detected by Bio-Rad XRS⁺ Imaging system.

2 × 2 × 2 mm³ tumor tissue explants were lysed group wise with 50 mM HEPES (pH 7.4), 100 mMNaCl, 0.1% CHAPS, 1 mM DTT, and 0.1 mM EDTA and protease inhibitor cocktail (Calbiochem) containing buffer to extract the total cellular protein. Protein concentration was determined by Bradford assay (BIO-RAD, Protein Assay, cat no-500-0006). 30 μg of total cellular protein from each group were electrophoresed on 10% SDS–polyacrylamide gels followed by their transfer onto nitrocellulose membranes. The membranes were blocked with 10% skimmed milk in Tris Buffered Saline with Tween 20 (TBST) for 2 h at room temperature, followed by overnight incubation at 4°C with p-AKT (Cell signaling Technology, cat no-4060), p-ERK and β-actin (MP Biomedicals, cat no-0869100) diluted 1:1,000 in 1% BSA in TBST. Blots are then washed with TBST followed by incubation with Goat Anti-Rabbit IgG Polyclonal Antibody (HRP) (KPL) and Peroxidase AffiniPure Rabbit Anti-Mouse IgG (H + L) (Jackson ImmunoResearch LABORATORIES, INC.) at a dilution of 1:5,000 in TBST. SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific™) was used for developing blots and the images were taken using XRS+ imaging system (Bio-Rad). Densitometry analysis of the blots was done using ImageJ software. Values of p-AKT and p-ERK were represented normalized to β-actin (33 (link)).