Bca protein assay kit

The intervention of lung tissue on ice for 10 min with lysate containing 10 mM PMSF for full lysis. The protein concentration of tissue lysates was determined using the BCA Protein Assay Kit (Generay, Shanghai, China). Each lane contains 30 μg of protein and is separated by standard immunoblotting according to standard protocols. Primary antibodies against JAK2 (1:1000), STAT3 (1:1000), pSTAT3 (1:1000) and horseradish peroxidase (HRP)-conjugated secondary antibodies were derived from Cell Signaling Technology, Inc (Boston, USA). Blots were visualized by ECL with GAPDH as the loading control. Western blots were quantified by the FluorChem E system.

Cells infected with lenti-RBM5 were seeded into six-well plates, and total protein was extracted after 72 h with CelLytic™ MEM Protein Extraction Kit (Sigma-Aldrich, USA) followed by quantified with bicinchoninic acid assay (BCA Protein Assay Kit, Generay, Shanghai, China). Then, 5 μg total protein were separated on SDS-polyacrylamide gel, transferred to nitrocellulose membranes, and incubated overnight with primary antibodies against Bcl-2, Bax, β-catenin, cyclin D1 (Santa Cruz Biotechnology, USA) and TCF, DKK1, GAPDH, Caspase-3, TNF-α (Abcam, USA) at 4 °C. After that, membranes were washed and incubated in horseradish peroxidase-conjugated secondary antibody for about 1 h at room temperature and target protein were determined with ECL kit (Pierce, USA).

For immunoblotting, cells treated with different compounds were collected at the indicated time and lysed with the RIPA lysis buffer (Beyotime, China). Quantification of the cell lysates were assayed with a BCA protein assay kit (Generay, China). 20 μg proteins were subjected to electrophoresis using 12% SDS-PAGE gels and transferred onto polyacrylamide difluoride (PVDF) membranes (Millipore, United States). After being blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween-20, membranes were incubated with individual primary antibodies (1:1000) at 4°C overnight followed by secondary antibody (1:5000) at room temperature for 1 h. The blots were detected by ECL Western blotting Detection System (Millipore, United States). The band density was measured by LabWorks Image Acquisition and Analysis Software (UVP, Upland, CA, United States) and normalized with band density of β-actin or GAPDH.

Arthrodial cartilage was used for protein extraction. After arthrodial cartilage collection, the arthrodial cartilage of rats was crushed using a mortar and pestle in liquid nitrogen and then lysed with RIPA buffer in the presence of 1% protease inhibitor cocktail (Roche, Basel, Switzerland). Protein from cell cultures was also extracted using RIPA buffer. The supernatant was collected after centrifugation at 12,000 g and 4°C for 30 min. Protein concentration was quantified with the BCA Protein Assay Kit (Generay, Shanghai, China). After being denatured in boiling for 5 min in SDS sample buffer, 40 μg of total protein was separated by 6%-15% SDS-PAGE, blotted onto PVDF membranes, and then probed with the following antibodies (Cell Signaling Technology, Danvers, MA, USA): monoclonal anti-TLR4 antibody, monoclonal anti-NF-κB p65 antibody, monoclonal anti-NF-κB phosphorylated p-65 (P-p65) antibody, and mouse anti-β-action antibody, conjugated to horseradish peroxidase, which were used as secondary antibodies. Protein bands were visualized by incubation with BeyoECL Plus (P0018, Beyotime, China) for 1 min and imaged by a Gel Image System (Tanon, 5200, China). Densitometry was performed by using the enhanced chemiluminescence (ECL) detection system (Thermo Scientific, Rockford, IL, USA).

Total proteins from brain tissues or cultured cells were lysed in RIPA buffer (Cell Signaling Technology, MA, USA) with 1 mM phenylmethanesulfonyl fluoride (PMSF) and protease inhibitor (MCE, NJ, USA). BCA protein assay kit (Generay Biotechnology, Shanghai, China) was used to quantified protein concentrations. Equal amount of protein was applied for SDS-PAGE electrophoresis and then transferred to PVDF membranes (Millipore, MA, USA). Membranes were blocked with 5% skim milk at room temperature for 1 h, then incubated with primary antibodies C3 (1:1000, Abcam), iNOS (1:1000, Abcam), S100A10 (1:1000, Abcam), Arg1 (1:1000, Cell Signaling Technology), suppressor of cytokine signaling 1 (SOCS1) (1:1000, Abcam) and β-actin (1:5000, Cell Signaling Technology) overnight at 4 °C. HRP-conjugated secondary antibodies were used for further incubation with the membranes for 120 min, and signals on blots were developed by the ECL reagents (Millipore, MA, USA) and were analyzed by ImageJ software.