X ray lm

Whole cell lysates and mouse cerebral cortex and hippocampal tissue homogenates were prepared in RIPA buffer (1 × PBS,1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 10 mM βglycerophosphate, 1 mM sodiumorthovanadate,10 mM NaF,1 mM phenylmethylsulfonyl uoride (PMSF), and1 × Roche Complete Mini Protease Inhibitor Cocktail (Roche, Indianapolis,IN). SDS-PAGE, transferring,and immunodetection were performed as previously described [31] . In brief, equal amounts of total protein extracts were fractionated by12% SDS-PAGE and electrically transferred onto a polyvinylidenedi uoride (PVDF) membrane. Primary antibodies and appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies were used to detect the designated proteins.The bound secondary antibodies on the PVDF membrane were reacted to the ECLdetection reagents (Santa Cruz, CA) and detected via exposing to X-ray lms (Kodak,USA).

Cellular proteins were extracted after treatment with a cell-lysis buffer which containing proteinase inhibitors on ice for 30 min and the samples were centrifuged at 12,000 rpm at 4℃ for 15 min. Then, the total protein was quanti ed using a BCA protein assay kit according to the manufacturer' instructions. The samples were subjected to 10% or 15% SDS-PAGE, and the protein bands were transferred to polyvinylidene di uoride (PVDF) membranes. After blocking with 5% dried nonfat milk, the membranes were incubated overnight at 4°C with an appropriate primary antibody. Then, the membranes were washed three times in Tris-buffered saline with tween 20 (TBST) and incubated for 1 h at room temperature with a secondary antibody conjugated to horseradish peroxidase. After three washes in TBST, the bands on the membranes were visualized with an ECL chemiluminescence reagent and X-ray lm (Kodak, Rochester, NY, USA) as described previously.

Generally, the lysed protein samples were subjected to SDS-PAGE and transferred onto nitrocellulose membranes by electrophoresis, while aliquots of samples were used to determine the protein concentration of each sample using a bicinchoninic acid (BCA) kit (KGPBCA, KeyGEN Biotech, Nanjing, China). Subsequently, membranes were incubated in blocking buffer for 2 h and incubated overnight at 4 ºC with the following primary antibodies: anti-TLR4 (1:200, SC-10741, Santa Cruz, CA, USA), anti-p-IKKα+b (1:500, ab2064, Abcam, Cambrige, UK), anti-IKKα+b (1:1000, ab178870, Abcam, Cambrige, UK), anti-p-IκBα (1:1000, ab12135, Abcam, Cambrige, UK), anti-IκBα (1:500, ab32518, Abcam, Cambrige, UK), anti-p-NF-κB p65(1:500, #3033, Cell signaling Technology, Boston, USA), anti-NF-κB p65 (1:1000, ab16502, Abcam, Cambrige, UK), and anti-HO-1(1:2000, ab13243, Abcam, Cambrige, UK). GAPDH (1:1500, ab8245, Abcam, Cambrige, UK) was used as a control on the same membranes. Applied secondary antibodies, the bands were detected with West Dura Extended Duration Substrate (Pierce, USA) and x-ray lm (Kodak, USA) and then analyzed by Bandscan 5.0 software via comparison with GAPDH.