Phosphatases inhibitors

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.

For western blot analysis HUVECs were lysed in Radioimmunoprecipitation assay buffer (Sigma-Aldrich) adding 10 % proteases and 1 % phosphatases inhibitors (Sigma-Aldrich Química, S.L., Madrid, Spain). Protein content was determined using Bradford assay buffer (Sigma-Aldrich Química, S.L., Madrid, Spain). Fifty micrograms of lysates were separated by electrophoresis using polyacrylamide gel electrophoresis gels (4–12 %; LonzaIbérica S.A.U., Barcelona, Spain) and transferred to a polyscreen polyvinylidene difluoride membrane (Perkin Elmer, Waltham, MA, USA). After blocking with 5 % non-fat dried milk or 5 % bovine serum albumin, membranes were incubated with the respective primary antibodies—cleaved caspase3 (Asp175) antibody (#9661s) and p21 Waf1/Cip1 (12D1) antibody (#2947; Cell Signalling Technology)—overnight at 4 °C. The membranes were then incubated with the appropriate secondary horseradish peroxidase-conjugated IgG antibodies (GE Healthcare Europe GmbH, Barcelona, Spain) at a 1:3,000 dilution for 1 h at room temperature. Blots were visualized with ECL reagent (Pierce Biotechnology, Rockford, IL, USA) using a LAS4000 Lumi-Imager (Fuji Photo Film, Valhalla, NY, USA). β-Actin—ACTB antibody (A-2066; Sigma-Aldrich Química, S.L., Madrid, Spain)—served as the loading control. Protein spots were quantitated with Image J software (http://rsb.info.nih.gov/ij/index.html).