Anti tlr4 antibody

CD14⁺ monocytes were lysed with whole cell lysis buffer and denatured at 100 °C for 5 min. Protein was then separated by SDS polyacrylamide gel electrophoresis and transferred onto PVDF membranes. The membranes were blocked in PBST buffer that contained 5% nonfat dry milk and were incubated overnight at 4 °C with an anti-TLR4 antibody (1:1000, Abcam, Cambridge, UK), anti-RFX1 antibody (1:1000, Genetex, Irvine, CA, USA), or anti-β-actin antibody (1:1000, Santa Cruz, Dallas, Texas, USA). The experiments were repeated three times, and the relative protein expression level was quantified with band density value normalized to β-actin.

RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd) was employed to extract protein samples from imDCs and HEK293T cells. A BCA Protein Assay Kit (Sangon Biotech, China) was employed to determine the concentration of extracted protein samples as previously described (Roman et al., 2020 ). Next, 25 µg protein samples were separated by SDS‐PAGE on 12% gels and transferred to PVDF membranes. After blocking, the membranes were incubated with primary antibodies, secondary antibodies, and ECL Western blotting substrate (Beijing Solarbio Science & Technology Co., Ltd). This was followed by visualizing using a Bio‐Rad ChemiDoc XRS⁺ System. The primary antibodies used in this experiment were as follows: anti‐myeloid differentiation primary response 88 (MyD88) antibody (1:5000; Abcam, UK), anti‐TLR4 antibody (1:300; Abcam), anti‐Casitas B‐cell lymphoma‐b (cbl‐b) antibody (1:500; Santa Cruz Biotechnology), anti‐Ubiquitin antibody (1:5000; Abcam), anti‐Myc tag antibody (1:500; Abcam), anti‐HA tag antibody (1:1000; Abcam), anti‐DDDDK tag (Binds to FLAG^® tag sequence) antibody (1:2000; Abcam), and anti‐GAPDH antibody (1:10,000; Abcam). GAPDH was used as a loading control.

The following materials were used: anti-Bcl-2 antibody (Abcam, 182,858), anti-Bax antibody (Abcam, 32,503), anti-GAPDH antibody (Abcam, 8245), anti-cleaved caspase-3 antibody (Cell Signaling Technology, 9664), anti-IL-1β antibody (Cell Signaling Technology,12,242), anti-IL-6 antibody (Cell Signaling Technology, 12,912), anti-IL-10 antibody (Abcam, 189,392), anti-TNF-α antibody (Abcam, 183,218), anti-TLR4 antibody (Abcam, 22,048), anti-MyD88 antibody (Abcam, 133,739), anti-NF-κB antibody (Abcam, 16,502), anti-p-NF-κB antibody (Abcam, 76,302), goat anti-rabbit HRP-conjugated antibody (Abcam 6721), and goat anti-mouse HRP-conjugated antibody (Abcam 6789).

Deparaffinized 4 μm sections were microwaved at 95°C for 20 min in 0.01 mol/L citric acid buffer (pH 6.0) and then incubated with 3% H₂O₂ for 15 min to consume endogenous peroxidase. After washing with PBS, the sections were incubated with anti‐TLR4 antibody (1:200; Abcam), anti‐MyD88 antibody (1:200, Boosen) and anti‐p65 antibody (1: 200; Cell Signaling Technology) for 2 h at 37°C. After washing with PBS, sections were incubated with HRP‐labelled goat anti‐rabbit secondary antibody (1: 1,000; Santa Cruz) for 30 min at 37°C. Finally, diaminobenzidine substrate (ZSGB‐BIO) was used to visualize positive areas.

Radix Aconiti Lateralis Preparata, Rhizoma Zingiberis, and Radix Glycyrrhizae were offered by the pharmaceutical department of Chinese herbal medicine of Guangdong Provincial Hospital of Chinese Medicine. Anti-TLR4 antibody (Cat. ab30667) was supplied by Abcam (USA). Total RNA Kit II was provided by Omega (USA). PrimeScript™ RT reagent kit was purchased from Takara Biotechnology Co., Ltd. (Dalian, China). The DNA ladder/marker (25-500 bp) was obtained from Shanghai Sangon Biological Engineering Co., Ltd. (Shanghai, China). PVDF membranes were purchased from Millipore (MA, USA). Rat tumor necrosis factor- (TNF-) α and interleukin- (IL-) 10 enzyme-linked immunosorbent assay (ELISA) kits were offered by Dakewe Biotech Company (Shenzhen, China). Rat Cortisol ELISA kits were obtained from BioVision (California, USA). Rat ACTH ELISA Kits were purchased from Cusabio Biotech Company (Wuhan, China).

Anti-phospho-p38 (Cat No. 9211), anti-phospho-p65 (Cat No. 3033), anti-p38 (Cat No. 9212), anti-p65 (Cat No. 8242), anti-S100A8 (Cat No. 9212), anti-CD8 (Cat No. 98941), anti-Cleaved-Caspase-3 (Cat No. 9661) antibodies, were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-TLR4 antibody (Cat No. ab13556), anti-Ki-67 (Cat No. ab16667) were purchased from Abcam (Cambridge, UK). Anti-S100A8 antibody were generated in our laboratory as described previously [8 (link)]. The purity of recombinant proteins was above 98% as judged by SDS-PAGE analysis. Endotoxin levels of recombinant proteins determined by LAL test (WAKO Chemicals, Japan) were <0.02 EU/µg. SW480 cell line was purchased from ATCC (Manassas, VA, USA), LLC cell line was purchased from RIKEN BioResource Research Center (Ibaraki, Japan) and MC38 cell line was kindly provided by Dr. Nicolas P. Restifo (NIH, Bethesda, MD). All cell lines were checked every two months, and found to be mycoplasma free.