Immature GIE stiffness is dependent on STEVOR S₃₂₄ phosphorylation. (A) Schematic representation of STEVOR recombinant protein overexpressed in the Full-Ty1, the Full_S324A-Ty1, and the Full_S324E-Ty1 lines. Red bar, The serine 324 in the Full-Ty1 line is mutated in alanine 324 in the Full_S324A-Ty1 line and in glutamate 324 in the Full_S324E-Ty1 line, respectively. (B) Western blot analysis of STEVOR expression in stage III GIE from the Full-Ty1, the Full_S324A-Ty1, the Full_S324E-Ty1 and the Null lines cultivated with (+BSD) or without (−BSD) blasticidin. Immunoblots were probed with a rabbit antibody directed against STEVOR (PFC0025c) and with a rat antibody directed against HSP70 to normalize expression. Quantitation of signal intensities was realized using Quantity One software (Bio-Rad). (C) Immunofluorescence analysis of stage III GIE from the Full-Ty1, the Full_S324A-Ty1, and the Full_S324E-Ty1 lines. Infected erythrocytes were stained with anti-Ty1 antibodies followed by anti-mouse Alexa 488–conjugated IgG. Pictures were taken under identical exposure conditions. The bars represent 2 µm. (D) Retention rates in microsphilters of stage III GIE from the control (light gray), the Full-Ty1, the Full_S324A-Ty1, the Full_S324E-Ty1, and the Null lines cultivated with (+BSD, dark gray) or without (−BSD, medium gray) BSD. Error bars denote the standard error of the mean. Outliers are shown as open circles. Stars represent highly significant differences in retention rates (***P < .001). ns, nonsignificant differences in retention rates.