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. 2019 May 10;9:158. doi: 10.3389/fcimb.2019.00158

Figure 2.

Figure 2

EspC bound to TLR4 but not TLR2. (A) Cell lysates were incubated with EspC (5 μg/mL) for 12 h and were then immunoprecipitated with anti-mouse IgG, anti-TLR2, or anti-TLR4 antibodies. The proteins were then visualized by western blotting with anti-HIS, anti-TLR2, or anti-TLR4 monoclonal antibodies. Total cell lysates were used as input control. Alternatively, cell lysates were incubated with EspC immobilized on Ni-NTA beads, and bead-bound proteins were loaded on gels for immunoblotting using anti-HIS, anti-TLR2, or anti-TLR4 monoclonal antibodies. (B) RAW264.7 cells were treated with EspC (5 μg/mL) for 30 min, fixed, stained with primary antibodies (mouse anti-HIS, rabbit anti-mouse TLR4, and rabbit anti-mouse TLR2 antibodies), and then re-incubated with appropriate Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies and DAPI. The cells were photographed under a fluorescent microscope (original magnification: 100 × ). Scale bar = 10 μm. Data are representative of three independent experiments. (C) Peritoneal macrophages derived from TLR4−/− mice or C57BL/6 mice were treated with EspC (5 μg/mL) for 30 min, fixed, stained with primary antibodies (mouse anti-HIS and rat anti-mouse TLR4 antibodies), and then re-incubated with appropriate Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies and DAPI. The cells were photographed under a fluorescent microscope (original magnification: 100 × ). Scale bar = 5 μm. Data are representative of three independent experiments.