Monoclonal rabbit anti tlr4

The homogenate of the small intestine was splitted with a lysis buffer, containing a cocktail of phosphatase and proteinase inhibitors and PMSF (Chauhan, 2019) (link). Following denaturation, the lysates were separated on a 10% SDS-PAGE gel and transferred to polyvinylidene difluoride PVDF membranes. The membranes blocked with 5% non-fat powdered milk in TBST (Tris-HCl buffered saline (PH8.8) containing 0.1% Tween20) for 1 h at room temperature and then incubated overnight at 4℃ with a monoclonal rabbit anti-TLR4 (1: 1000, Proteintech Group), anti-TLR2 (1: 1000, Proteintech Group), or anti-GAPDH (1: 5000, Proteintech Group) primary antibody. After washing in TBST, the membrane was incubated for 1 h at RT with a HRP-conjugated goat anti-rabbit antibody (1:1000; Jackson Immunoresearch Laboratories, PA, USA), and the protein bands were visualized using Enhanced Chemiluminescence Kit (Thermo, Production batch: PICPI23223). Images of the protein bands were recorded using the Image Quant LAS 4000 system (Tanon-5200, Tanon, USA), and the band intensities were quantified using Imagequant TL software (version 2.2.4, Tanon, USA). The density of proteins was quantified by Quantity One (Thermo, USA). Three mice in each group were randomly selected for Western blotting.