Emm antigen is carried by the free GPI. (A) Cell surface expression of the Emm antigen and CD59 in K562 WT and K562 PIGA KO cells. The gray profile corresponds to K562 cells incubated with only the secondary antibody. (B) Cell surface expression of the Emm antigen and CD59 in K562 WT and K562 PIGS KO cells. The gray profile corresponds to K562 cells incubated with only the secondary antibody. (C) Immunofluorescence analysis of the Emm antigen. Fixed and permeabilized cells were stained with an anti-Emm antibody (green) and 4′,6-diamidino-2-phenylindole (DAPI; blue). (D) Western blot analysis of Emm and CD59 expression in K562 WT, K562 PIGA KO, and K562 PIGS KO cells. Twenty micrograms of protein from cell lysates was resolved by SDS–polyacrylamide gel electrophoresis under reducing conditions and probed with anti-Emm and anti-CD59 antibodies. Actin was used as a loading control. (E) Surface level analysis of Emm antigen on RBCs after incubation with brain polar lipids. Control RBCs (left panel) or Emm-negative RBCs (right panel) were incubated in 1% methanol in PBS containing 0.5 mg of brain polar lipids (black dotted lines) or with only 1% methanol in PBS (blue dotted lines) for 120 minutes. Emm antigen adsorption on the RBC surface was monitored by fluorescence-activated cell sorting using the anti-Emm antibody.