Anti-Emm did not recognize the second EtNP on the GPI-APs. (A) Schematic representation of the free GPI and GPI-AP structures in K562 WT and K562 PGAP5-deficient cells (MPPE1 KO) cells. (B) Cell surface expression of the Emm antigen and CD59 in K562 WT and K562 MPPE1 KO cells. The gray profile corresponds to K562 cells incubated with only the secondary antibody. (C) Immunofluorescence analysis of Emm antigen. Fixed and permeabilized cells were stained with the anti-Emm antibody and 4′,6-diamidino-2-phenylindole (DAPI). (D) Western blot analysis of Emm and CD59 expression in K562 WT, K562 PIGG KO, and K562 MPPE1 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) CD59 was immunoprecipitated from the control RBC membrane (Ctrl) or Emm-negative RBC membrane (Emm-). Input and immune complexes (IP CD59) were analyzed by SDS–polyacrylamide gel electrophoresis under reducing conditions with heat denaturation, followed by western blot analyses using anti-Emm and anti-CD59 antibodies. Actin was used as a loading control.