Figure 5.

Differences of Band 3 Y²¹ (A, C) and Y³⁵⁹ (B, D) phosphorylation according to P. falciparum infection and abnormal hemoglobin S carriage. 4-12% gradient gels were loaded with 20 µg/lane of ghost protein extracts from 2 HbAA (HbAA1 and HbAA2) and 2 HbAS (HbAS1 and HbAS2) donors. After separation of erythrocyte ghost lysate proteins by SDS-PAGE and transfer on nitrocellulose, tyrosine (Y)-phosphorylation was analyzed using anti-phosphoY21 (A) and anti-phosphoY359 (B) Band 3 antibodies. Tyrosine phosphorylation intensities were measured with Image Lab software, and these intensities were normalized to Band 3 quantity detected on the same membrane by western blot. For each sample, the phosphorylation intensity value of infected condition was reported to the non-infected condition intensity value (C, D). Independent western blots were realized twice, and technical replicate values for each point were loaded on the graph. Phosphorylation fold intensities for HbAA and HbAS samples were represented in blue and red respectively, with non-infected (circles) and infected (square) ghosts. Paired t-test (*p-value < 0.05; **p-value < 0.01, and ***p-value < 0.001) were performed to compare intensities from the same genotype group (HbAA or HbAS) donors. Unpaired Mann-Whitney t-test (*p-value < 0.05; **p-value < 0.01, and ***p-value < 0.001) was used to compare intensities from infected HbAA and infected HbAS donors. i, infected; ni, non-infected; ns, not significant.