Detergent compatible protein assay reagent

Cell lysates were prepared by scraping the T24 and J82 cells off the plate after treatment with the EzRIPA lysis kit (ATTO Corporation). The lysates were transferred into microcentrifuge tubes and centrifuged at 15,000 × g for 15 min at 4°C. After transferring the supernatant into a new tube, the protein concentration was measured using a detergent-compatible protein assay reagent (Bio-Rad Laboratories, Inc.). Gel electrophoresis, membrane transfer and blocking, and antibody binding were performed as described previously (28 (link),32 (link)). Proteins (25 µg protein/lane) were loaded and separated on 10 or 15% SDS-PAGE gels. The proteins were transferred to polyvinylidene difluoride membranes, and blocked with 5% skim milk for 1 h at room temperature. The membranes were incubated with the aforementioned primary antibodies overnight at 4°C. After washed by PBST (0.05% Tween-20), the membranes were incubated with the aforementioned secondary antibodies at room temperature for 2 h. Chemiluminescence was detected using Luminata Forte Western HRP Substrate (Merck Millipore) by ImageQuant LAS 40000 mini (GE Healthcare). Membranes were reprobed with GAPDH antibody as an internal loading control.

The liver of mice was dissected 1 h and 3 h after sepsis induction. After dissection from hippocampus and liver, tissue was lysis with SDS lysis buffer containing 0.1 mM Na3VO4, and 20 mM NaF. After brief sonication to shear DNA and reduce viscosity, the concentration of protein was determined with the detergent-compatible protein assay reagent (Bio-Rad Laboratories, USA) using bovine serum albumin as the standard. The western blot analysis was performed according to our previous study [38 (link)42 (link)]. The membranes were then exposed to a Luminescent Image Analyzer (LAS-4000, Fuji Film Co., Japan) for the detection of light emission. Specific signals were quantified with the Multi-Gauge Version 3.1 (Fuji Film) and expressed as a percentage of the control.