CHO cells coexpressing the A histo-blood group antigen and the SARS-CoV S protein. (A) Flow cytometry analysis of the expression of A antigen and the S protein–EFGP construct on CHO mock-transfected cells (mock), double transfectants either with the Fut2 and S protein constructs (Fut2/SP) or the Fut2 and A enzyme cDNAs (Fut2/A), triple transfectants with the Fut2, A enzyme and S protein constructs (Fut2/A/SP). Fluorescence of the S–EFGP molecule was directly recorded on the FL1 channel. Detection of the A antigen was performed using an anti-A mAb followed by Cy5-labeled anti-mouse IgG and recorded on the FL4 channel. (B) Confocal microscopy analysis of the A antigen (A TRITC) and the S protein–EFGP construct coexpression on CHO cells triple transfectants. Detection of the A antigen was performed using an anti-A mAb followed by TRITC-labeled anti-mouse IgG. (C) Western blot analysis of transfected CHO cells glycoproteins. Total protein extracts from CHO Fut2 simple transfectants, CHO Fut2/A double transfectants, and CHO Fut2/A/SP triple transfectants were submitted to SDS–PAGE and Western blotting. Glycoproteins carrying A histo-blood group epitopes were detected with an anti-A mAb. The arrow shows the expected molecular size of the EGFP–SP fusion protein.