Treatment of chicken ART4 transfected C-33A cells with PI-PLC and PNGase F. A) C-33A cells (4 × 107/ml) transfected with the chicken ART4 containing plasmid or the empty plasmid (pSecTag B) were stained with the anti-Flag antibody (black bar) or the isotype control (white bar) after being incubated for 1 h at 37°C in the presence or absence of PI-PLC (5 U/ml). Data show the mean of % positive cells ± S.E.M. of three experiments. ** p = 0.007 (Student's t-test) B) Western blot analysis using the anti-Flag antibody was performed with supernatants of chicken ART4 transfected C-33A cells after incubation in the presence or absence of PI-PLC. Data show one representative experiment out of three. C) The supernatant of chicken ART4 transfected cells was incubated for 1 h in the presence or absence of PNGase F (20000 U/ml) at 37°C. PNGase F alone was run as a control. After boiling the samples in SDS-PAGE sample buffer, Western blot analysis using the anti-Flag antibody were carried out. The membrane was stained with Ponceau S (D). Similar results were obtained in three separate experiments.