Fig. 2

rA2 binds to and reduces TLR4 membrane expression in neutrophils. A Representative gel images of Co-IP coupled with Western blotting analysis of the binding of AXNA2 to TLR4 in the membrane of neutrophils, which were treated with PBS or rA2 (2 μg/mL) for 1 h. Anti-TLR4 antibodies were used for immunoprecipitation, and the immunoprecipitates and membrane proteins were analyzed by immunoblotting with anti-AXNA2 antibodies. Na⁺K⁺ATPase served as an equal loading control of cell membrane protein. B Representative confocal microscope images of co-immunostained with AXNA2 and TLR4 in neutrophils, which were treated with PBS or rA2 (2 μg/mL) for 1 h. The DAPI was used to stain the nucleus. Scale bar, 50 μm. The scale bar in the yellow box is 10 μm. C Representative flow cytometry plots for TLR4⁺ or TLR2⁺ immune cells obtained from blood samples of TBI at 24 h. D Quantitation of TLR4 and TLR2 expressing in periphery neutrophils, monocytes, and T lymphocytes from sham, TBI, and TBI + rA2 mice, n = 4. Data are expressed as mean ± SEM; *p < 0.05 compared to sham; ^#p < 0.05 compared to TBI