Bca protein assays kit

5 ml blood was taken from inferior vena cava after rats subjected to I/R 90 min/3 h, then centrifuged at 3500 rpm, 4 °C for 10 min, and the plasma was stored in − 80 °C refrigerator for later use. The rat was decapitated and the infarcted hemisphere was rapidly freezed with liquid nitrogen and stored in − 80 °C refrigerator for later use. After balance to room temperature, the infarcted hemisphere was grinded with high throughput tissue lapping instrument (CK1000D, Thmorganh). 500 μl PMSF: RIPA (1:100) lysis buffer and 1 μl protease inhibitor was added to 100 mg rats brain homogenates. The mixture was re-grinded to thoroughly blended, and then centrifuged at 14000 rpm, 4 °C for 10 min, and the supernatant was used for determination of protein concentration by BCA Protein Assays kit (Thermo Fisher Scientific, USA) according to the manufactures protocol by a fixed investigator who was blind to the groups. Interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) in plasma and brain homogenates was measured by ELISA kit (Invitrogen, Carlsbad, CA, USA) according to the manufactures protocol.

Cells were lysed with buffer containing 0.5% SDS, 5% mercaptoethanol, and 1% protease inhibitor mixture. Protein concentration was determined using a BCA protein assays kit (23225, Thermo Scientific). Proteins were subjected to SDS-PAGE and then transferred to a polyvinylidene fluoride membrane. After blocking with 5% BSA for 2 h at room temperature, the membranes were, respectively, incubated with the indicated antibodies overnight at 4 °C. The dilution ratios of primary antibodies are shown in Table S1. After incubation with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature, protein expression was detected by the enhanced chemiluminescent method and imaged with a Molecular Imager System (Bio-Rad, USA).

Firstly, the RIPA lysis buffer (Beyotime;Shanghai, China) was applied to extract the total protein from the cell precipitation, and its concentration was measured using BCA protein Assays Kit (Thermo Fisher Scientific; Shanghai, China). Equal amount of proteins (20 μg) was separated on SDS-PAGE gel, and then transferred onto polyvinylidene fluoride (PVDF) membranes. Subsequently, the membranes contained proteins were incubated with 5% BSA, the specific primary and secondary antibody respectively. Finally, the protein bands were visualized by GeneSnap using SynGene system.

The extraction of HCC cell total proteins was acquired using RIPA lysis buffer (Cell Signaling Technology, MA USA) containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). Protein concentration was determined by the BCA Protein Assays kit (ThermoFisher, MA USA). Lysates were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and electro-transferred to a polyvinylidene difluoride membrane (Bio-Rad, CA USA) that was blocked with 5% skimmed milk for 1 hour at room temperature and cultured overnight at 4°C with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Abcam, MA USA). Immunodetection was performed using chemiluminescence detection system (Bio-Rad, CA USA). Antibodies include HMGB1, (1:1000, from Proteintech, IL USA), PARP, cleaved-PARP (1:1000, from Abcam, MA USA), NF-κB-p65, phosphor-NF-κB-p65 (1:1000, from CST, MA USA), and HIF-1α (1:1000, from BD Biosciences, CA USA); β-actin was an internal control.

Cells were seeded in 6-well plates for pre-treatment (miRNA mimics and siRNAs). After treatment, the cells were lysed with RIPA lysis buffer (Thermo Fisher Scientific, Massachusetts, USA) containing Complete Mini protease inhibitor cocktail and PhosSTOP phosphatase inhibitor (Roche Applied Science, Penzberg, Germany). The protein concentrations of the samples were determined by the BCA Protein Assays Kit (Thermo Fisher Scientific, Massachusetts, USA) and quantified with a GloMax microplate reader (Promega GmbH, Walldorf, Germany).
The primary and secondary antibodies used in this study are listed in Additional file 2: Supplementary Table 4. The membranes were scanned and probed using the Odyssey Infrared Imaging System (LI-COR Biosciences, Nebraska, USA). The signal intensity of the band was quantified by using ImageStudio software and median background subtraction (LI-COR Biosciences, Nebraska, USA).