Mouse anti human tlr4

U-87MG-Sh, U-87MG-NC and U-87MG cells were collected in the logarithmic phase of growth and washed with cold PBS twice. Total protein was collected by adding cell lysis solution (RIPA:PMSF, 100:1; Beijing Solarbio Science & Technology Co., Ltd.). The protein concentration was evaluated via bicinchoninic acid assay. Protein (50 µg/lane) was separated via 10% SDS-PAGE then transferred to PVDF membranes before being blocked overnight at 4°C with 5% skimmed milk. Next, rabbit-anti-human β-actin (1:4,000; cat. no. ab8227; Abcam) or mouse-anti-human TLR4 (1:500; cat. no. ab22048; Abcam) monoclonal antibodies were added for incubation at room temperature for 1 h. The membrane was washed three times, then incubated at room temperature for 2 h with horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit secondary antibodies (1:20,000; cat. nos. ab205719 and ab205718, respectively; Abcam). The antigen-antibody reaction was visualized by detection using an ECL assay (EMD Millipore). Relative expression levels of TLR4 protein were normalized to those of β-actin using Odyssey v3.0 software (LI-OR Biosciences). A total of three independent repeats were performed.

Total cellular protein was extracted from cultured PDL-CD105⁺ cell populations using the RIPA buffer (Thermo Scientific, Rockford, IL, USA) supplemented with protease inhibitor cocktail (Sigma, P2714), following the manufacturer’s specifications. Protein concentration was measured by the Bradford method (Bio-Rad Laboratories, Benicia, CA, USA) with bovine serum albumin (BSA) as a standard, and was measured spectrophotometrically at 595 nm. Equal amounts of protein per sample were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) 10% and transferred to a nitrocellulose membrane (Amersham™ Hybond ECL, Amersham BioSciences™, GE Healthcare, Little Chalfont, Buckinghamshire, UK). The membranes were blocked with 3% BSA in TBS for 1h at room temperature, incubated overnight with a 1:1000 dilution of primary antibody mouse anti-human TLR4 (Abcam), and then incubated with peroxidase-conjugated secondary antibodies (Anti-mouse 1:2500) (Santa Cruz Biotechnology, Dallas, TX, USA) for 2 h. The membrane was developed using an ECL reagent (SuperSignal West Femto Substrate, Thermo Scientific) and the signals were detected using radiographic films (Kodak, Rochester, NY, USA).