Erythrocyte Ghost Preparation and Protein Extraction

HbAA and HbAS erythrocyte ghosts (RBC membranes and cytoskeleton) were obtained by hypotonic lysis at 4°C of either infected or non-infected erythrocytes according to a protocol from Azouzi et al. (2015) (link). The RBCs were first washed three times in PBS 1X and then incubated with 10 volumes of lysis buffer (5 mM Na₂HPO₄, 0.35 mM EDTA, pH 8.0 with proteases (Roche Diagnostics) and phosphatases inhibitors (Sigma-Aldrich)) for 5 minutes on ice. Solutions were afterwards centrifuged at 50,000 x g for 20 minutes at 4°C. For the infected samples, the ghost layer was collected in a new tube to separate ghosts from parasite pellet. The reddish supernatant (containing hemoglobin) was removed, and the ghosts and parasites were washed in lysis buffer until the pellets became colorless. Protein extraction from erythrocyte membranes and parasites was performed by adding one volume of extraction buffer 3X (3% NP40 (Sigma-Aldrich), 3% SDS (Fluka), 90 mM Tris pH 8, 3 mM EDTA, pH 8 with proteases (Roche Diagnostics) and phosphatases inhibitors (Sigma-Aldrich)) to two volumes of each pellet. For parasite extracts, a supplementary sonication step was realized to solubilize the proteins. The protein content was measured by BCA (bi-cinchoninic acid) assay (Micro BCA™ Protein Assay Kit, ThermoFischer Scientific). The preparations were finally stored at -80°C until analysis.