Figure 5: CD36 cooperates with TLR4 during E. coli internalization and phagocytosis.

(A,B) Cells were transfected with pBiFC-VC155-TLR4 (A, left upper) or pBiFC-VN155-CD36 (B, right upper) alone, and then Hoechst 33342-labeled E. coli was added for 2 h at 37 °C. (C) Hoechst 33342-stained E. coli bacteria were used to stimulate cotransfection of VC-155-TLR4 and VN-155-CD36 cells for 2 h. (D) pGMECs were cotransfected with pBiFC-VC155-TLR4 and pBiFC-VN155-CD36 plasmids, and the cells were stained with Hoechst 33342 to visualize the nuclei. (E) pGMECs were cotransfected with pBiFC-VC155-TLR4 and pBiFC-VN155-CD36 plasmids, and then the cells were stimulated with LPS (10 μg/ml) and Hoechst 33342 for 2 h. The images are representative of multiple fields from three experiments. Scale bar: 10 μm. (F) Cells were transiently cotransfected with pef-NEO-Flag-CD36 and pef-NEO-Myc-TLR4 and then incubated with LPS for 12 h or with E. coli for 2 h. The cell lysates (input) were probed for Flag-CD36, Myc-TLR4, and β-actin. (G) Flag-CD36 was immunoprecipitated from the cell lysates using mouse anti-Flag antibody. (H) The cells were lysed and plated on agar after incubation with E. coli (10⁷ cfu/ml) in pGMEC manipulated with mock, Ad-CD36, or si-CD36 for 2 h. Equal protein amounts were immunoprecipitated (IP: Flag-CD36) using anti-Flag antibody. All data are presented as the mean ± SEM from three experiments. *P < 0.05, **P < 0.01, and not significant (NS).