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500 ng recombinant human TLR4 (hTLR4) (Sino Biological Inc., Beijing, China) was coated onto 96-well immune plates (Thermo Fisher scientific), and nonspecific binding was blocked with 10 mM HEPES buffer (pH 7.4) containing 0.15 M NaCl, 5 mM CaCl 2 , and 2% fatty acid-free BSA (buffer A)

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Fifth, since we utilized non-time-series data, further investigation is required to understand specific temporal variations