Horseradish peroxidase hrp conjugated goat anti mouse rabbit igg

Total proteins were isolated from the NP cells using RIPA buffer (Cell Signaling Technology, Inc.). The BCA Protein Assay kit (Pierce) was applied to measure the protein concentration, which was adjusted to a concentration of 6 µg/µl using 1X loading buffer and DEPC water. Electrophoresis was used to separate the samples using a 10% SDS-PAGE gel followed by transfer onto a polyvinylidene fluoride membrane (PVDF, Millipore). After blocking in 5% non-fat milk in PBST [0.1% Tween-20 in phosphate-buffered saline (PBS)] for 1 h, the membrane was incubated with the primary antibody overnight at 4°C, and subsequently incubated with secondary antibody [horseradish peroxidase (HRP)-conjugated goat anti-mouse/rabbit IgG, 1:2,000; sc-516102/sc-2357; Santa Cruz Biotechnology, Inc.] at room temperature for 2 h. Developer (EZ-ECL kit; Biological Industries BI) was used for development, and the gray value of the strips were analyzed and quantified using imageJ software (version 5.0; Bio-Rad). The antibodies utilized included anti-GAPDH (mouse; 1:1,000; LS-B1625; LifeSpan BioSciences, Inc.), anti-TLR4 (rabbit; 1:500; ab13556; Abcam), anti-IκBα (mouse; 1:1,000; #4814), anti-p-IκBα (mouse; 1:1,000; #9246), anti-p65 (rabbit; 1:1,000; #8242), anti-p-p65 (rabbit; 1:1,000; #3039) and anti-TIMP3 (rabbit; 1:1,000; #5673) (all from Cell Signaling Technology, Inc.).