Fig. 2.
γ-PGA induced activation of macrophage was mediated by TLR4. BMDMs (1 × 106 cells/ml) isolated from TLR4-defective (C3H/HeJ), TLR2-deficient mice, or wild-type (C3H/HeN, C57BL/6) mice were stimulated with 100 ng/ml LPS or 1 mg/ml γ-PGA for 24 h. Concentration of TNF-α (a) and IP-10 (b) in the culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). RAW264.7 cells, murine macrophage cell line, were transiently transfected with siRNA targeting the coding regions of TLR4 (siTLR4) and the control siRNA. The mRNA and protein expression levels of TLR4 of siRNA transfected cells were determined by RT-PCR, fluorescence microscopy (×200) and flow cytometry analysis (c). The RAW264.7 cells transfected with siRNA were treated with 100 ng/ml LPS and 1 mg/ml γ-PGA. The supernatants were collected 24 h later and concentrations of TNF-α of siTLR4- or the control siRNA-transfected cells were determined by ELISA (d). Asterisks represent not detected. The results are expressed as mean ± SE of three independent experiments