Western blotting
Tissues and cells were homogenized with RIPA buffer containing PMSF.
Proteins were resolved on 10% SDS-PAGE and transferred to the PVDF
membrane. After blocking for 1 hr, membranes were incubated with primary
antibody TLR4 (1:100; Santa Cruz, USA, cat: sc-293072), phospho-NFκB p65
(1:800; CST, USA, cat: #3033), β-actin (1:1000; HuaBio, China, cat:
ET1701-80), and TATA-binding protein (TBP) (1:1000; HuaBio, China, cat:
HA500518). Membranes were subsequently incubated with peroxide-labeled
secondary antibodies. Immune complexes were detected using enhanced
chemiluminescence (Millipore, USA), and results were recorded using a
chemiluminescence imaging system (Biorad, USA).