The Emm antigen is composed of the second and third EtNPs of the free GPI. (A) Schematic representation of the free GPI in the plasma membrane. PIGN, PIGG, and PIGO catalyze the addition of EtNPs on the first, second, and third mannose, respectively. (B) Cell surface expression of the Emm antigen in K562 WT and K562 PIGN KO cells. The gray profile corresponds to K562 cells incubated with only the secondary antibody. (C) Cell surface expression of the Emm antigen in K562 WT and K562 PIGO KO cells. The gray profile corresponds to K562 cells incubated with only the secondary antibody. (D) Western blot analysis of Emm and CD59 expression in K562 WT, K562 PIGG KO, K562 PIGA KO, K562 PIGN KO, and K562 PIGO 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.