IF analysis of TLR4 and Frizzled 9. (A) Confocal microscopy analysis evidenced the coexpression of TLR4 and Frizzled 9 on neuronal cells (arrows) either in SM- than BM-treated samples. (B) Co-immunoprecipitation was performed on total protein extracts from ENSc cultured for 7 days in SM or BM. Immunoaffinity purification was carried out using goat anti-rat Frizzled 9 and rabbit anti-rat Wnt3a antibodies pre-immobilized onto Protein A Sepharose. Western blot analysis was assessed using 4%–15% gradient precast polyacrylamide gel. (C) By immunofluorescence assay, ENSc showed cytoplasmic vesicles immunoreactive (arrows) to rabbit anti-rat Wnt3a antibody. Bar: 15 μm.