Mouse monoclonal anti tlr4

Total protein was obtained from heart and lung tissues by sonication, centrifugation, and heat denaturation. The protein lysates were electrophoresed and separated on 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred onto nitrocellulose membranes (Millipore, Inc, USA). The membranes were blocked with 5% skim milk at room temperature for 1 h and then incubated overnight at 4°C with primary antibodies, including rabbit polyclonal Anti-Collagen I (1:800, ABcam, Inc.), mouse monoclonal anti-TGF-β1 (1:500, Abcam, Inc.), rabbit monoclonal anti-Smad3 (phosphor C25A9) (1:500, Cell Signaling Technology, Inc.), rabbit polyclonal anti-TNF-α (1:1000, Abcam, Inc.), mouse monoclonal anti-TLR4 (1:2000, Abcam, Inc.), and rabbit anti-NF-κB p65 (1:800; Santa Cruz, Inc.). The membranes were then incubated with the secondary antibody (1:2000) at room temperature for 2 h. Electrochemiluminescence was induced and the Gene Gnome Gel Imaging System (Syngene Co.) was used to capture the resulting images. Image J (NIH image, Bethesda, MD) was used to analyze the gel images. The results were expressed as density values normalized to GAPDH.

Cellular proteins were extracted from the primary astrocytes cells using RIPA buffer. The protein concentration in the supernatant fluid of the lysate was measured using a BCA kit. Proteins (50 µg) in the cell extracts were denatured with sodium dodecyl sulfate (SDS) sample buffer and separated using 10% SDS-polyacrylamide gel electrophoresis (PAGE). The proteins were transferred to a polyvinylidene fluoride (PVDF) microporous membrane (Millipore, Bedford, MA, USA), which was then blocked with 5% skim milk for one hour at room temperature. The membrane was incubated with primary antibody overnight at 4°C. The following primary anti-bodies were used: mouse monoclonal anti-TLR4 (Abcam, number: 22048, 1:1000), rabbit monoclonal anti-GFAP (CST, number: #12389p, 1:1000), mouse monoclonal anti-GAPDH (Bioworld Technology lnc. number: Ap0063, 1:1000). After adding the anti-rabbit or anti-mouse secondary antibody (Jackson Immuno Research Laboratories INC. number: 122825 and 122627, 1:1000) for 1 hour, the protein bands on the membranes were detected with an enhanced chemiluminescence kit.