Toll like receptor 4 tlr4

After the treated C28/I2 cells were harvested, 30 μg of protein was extracted by RIPA lysis buffer (Beyotime, Shanghai, China), separated on SDS-PAGE gel and then transferred to polyvinylidene difluoride (PVDF, Millipore, Bedford, MA, USA) membranes. Subsequently, the membranes were immersed in 5% nonfat milk for 2 h and incubated with primary antibodies against B cell lymphoma-2 (Bcl-2, 1:1,000, Abcam, Cambridge, MA, USA), Bcl-2-associated X protein (Bax, 1:2,000, Abcam), SOX11 (1:2,000, Abcam), phosphorylation-P65 (p-P65, 1:1,000, Thermo Fisher Scientific), P65 (5 µg/mL, Thermo Fisher Scientific), IkB-α (1:2,000, Abcam), p-IkB-α (1:3,000, Abcam), Toll-like receptor 4 (TLR4, 1:1,000, Abcam), or GAPDH (1:3,000, Abcam) at 4°C overnight. The membranes were then probed with a secondary antibody conjugated with horseradish peroxidase (1:5,000, Abcam) for 1 h at 37°C. The blots were visualized by RapidStep ECL Reagent (Millipore Corp., Billerica, MA, United States), and the results were observed using Bio-Rad ChemiDoc XRS + Chemiluminescence Imaging System.

The primary antibodies, including indoleamine 2, 3 dioxygenase1 (IDO1), ionized calcium binding adapter molecule 1 (Iba1), glial fibrillary acidic protein (GFAP), T-brain gene-2 (Tbr2), doublecortin (DCX), Prox1, act-casp3, PV, c-Fos, Calretinin, occluding, β-actin, toll-like receptor 4 (TLR4), and NF-κB, were purchased from Abcam, Invitrogen, Santa Cruz Biotechnology, and Cell Signaling Technology companies. The western blot and immunofluorescence procedures were performed following our previous protocols with minor modifications 26 (link). The modification is mainly the adjustment of the antibody dilution.