Fig 1: RBC cytoskeleton proteins are target substrates of the EV-20S proteasome.A Degradation of cytoskeletal proteins following Pf-derived EV treatment. Western blot analysis of naïve RBCs treated with Pf-derived EVs in the presence or absence of the proteasome inhibitor bortezomib. Commercial antibodies were used against five cytoskeletal proteins: ß-adducin (ADD2), erythrocyte membrane protein band 4.1 (EPB4.1), ankyrin-1 (ANK1), dematin (DMTN), and spectrin a-chain (SPTA1). For each gel, GAPDH was used as a loading control. B Quantification and averaging of three independent experiments (as in A). Each protein’s value was divided by the control value for that batch, thus creating a pair of ratios for each batch. The ratios of Pf-derived EVs were tested against the ratios of the Pf-derived EVs + bortezomib using a paired two-way t-test, error bars represent SD (DMTN *p = 0.0102, ADD2 *p = 0.01339, EPB41 *p = 0.02769, ANK *p = 0.01339). C Graphical illustration of the disorder prediction of human ß-adducin, Ebp4.1, ankyrin-1, dematin, and spectrin a-chain, generated by D2P2 (website: http://d2p2.pro), a database of protein disorder predictions. The level of disorder prediction is shown as a color intensity in which green segments represent disordered regions. The position of the unique phosphosites following treatment with Pf-derived EVs is indicated by orange circles. D Degradation assay of ghost naïve RBCs with purified 20S proteasome complexes. Bortezomib was used as a control for 20S proteasome inhibition. Panels display immunoblots using the relevant antibodies. Results represent three independent experiments (Supplementary Fig. S16). E Degradation of cytoskeletal proteins following Pf-derived EV treatment in the presence of kinase inhibitors mixture. Western blot analysis for naïve RBCs that were treated with Pf-derived EVs in the presence or absence of kinase inhibitor mixture (staurosporine and dihydrochloride). Antibodies were used against ankyrin-1 and dematin. GAPDH was used as a loading control (Supplementary Fig. S17). F Quantification and averaging of three independent experiments (as in E), using a paired two-way t-test, error bars represent SD (ANK *p = 0.0421, DMTN *p = 0.0402). Source data for A and D are provided in the source data file. Source data for B and F are provided in Supplementary Fig. S14.
Supplier Page from Abcam for Anti-Adducin 2 antibody