Ab203294

Cells or tissues were harvested at indicated times and homogenized in ice-cold suspension buffer supplemented with a proteinase inhibitor cocktail (Sigma-Aldrich). Protein concentrations were determined using the BCA Protein assay kit (Thermo Scientific, Waltham, MA). Equal amounts of protein were fractionated by SDS polyacrylamide gels, followed by immunoblotting with the following primary antibodies: NF-κB Pathway Sampler Kit (diluted at 1:1000, 9936, CST, MA), phospho-MAPK Family Antibody Sampler Kit (diluted at 1:1000, 9910, CST, MA), MAPK Family Antibody Sampler Kit (diluted at 1:1000, 9926, CST, MA), phospho-Stat6 antibody (Rabbit polyclonal, diluted at 1:1000, ab28829, Abcam), Stat6 antibody (Rabbit polyclonal, diluted at 1:1000, ab44718, Abcam), TRAF6 antibody (Rabbit polyclonal, diluted at 1:1000, ab94720, Abcam), TLR4 antibody (Rabbit polyclonal, diluted at 1:1000, ab13556, Abcam), CD14 antibody (Rabbit polyclonal, diluted at 1:1000, ab203294, Abcam), Myd88 antibody (Rabbit polyclonal, diluted at 1:1000, ab2064, Abcam), PPARγ antibody (Rabbit monoclonal (C26H12), diluted at 1:1000, sc-7273, Santa Cruz Biotechnology). Membranes were then incubated with peroxidase-conjugated secondary antibody, and specific bands were detected with a Bio-Rad (Hercules, CA) imaging system. Original blots were provided in Supplementary Fig. 8.

The RIPA lysis buffer, in addition to 1% protease inhibitor cocktail and 1% PMSF, was used to homogenize samples of the cerebral cortex. Following the addition of sodium dodecyl sulfate (SDS) loading buffer, the samples were boiled for 5 min, and proteins were subsequently detected by western blot analysis. The proteins were transferred to a polyvinylidene difluoride (PVDF) membrane after separation. A wide range of protein markers was run in parallel to detect the molecular weight of proteins. Skimmed milk was used for membrane blockage to reduce nonspecific binding. Proteins were probed with anti-TLR4 (1:1000, ab13867; Abcam, Cambridge, UK), anti-CD14 (1:500, ab203294; Abcam), anti-IRAK1 (1:1000, no. 4504; Cell Signaling Technology, Danvers, MA, USA), anti-p65 (1:1000, no. 8242; Cell Signaling Technology), anti-phospho-p65 (1:1000, no. 3033; Cell Signaling Technology), and anti-β-actin (1:1000, abs119600; Abcam). The data were quantified using the Image Studio Lite ver. 5.2 software.