Man4 inhibits detection of free GPIs with T5 mAb.
A, flow cytometry of 3BT5 cells transiently transfected with empty vector (blue) or PIGZ cDNA (orange). Top, staining with T5 mAb; bottom, staining with anti-CD59. Dotted lines, secondary antibody only. B, Western blotting of free GPI in the lysates of 3BT5 cells transiently transfected with empty vector (left) or PIGZ cDNA (right). DAF, a GPI-AP; TfR, a loading control. C, flow cytometry of free GPIs on 3BT5-PIGZ KO (orange) and 3BT5 (blue) cells. Cells were stained with T5 mAb and Alexa Fluor 488 secondary antibody. Dotted line, 3BT5 stained by secondary antibody only. D, Western blotting of free GPIs in the lysates of 3BT5 (left) and 3BT5-PIGZ KO (right) cells. Similar results were obtained in at least two independent experiments.