Figure 2

Confirmation of chimeric protein expression and biotinylation activity. (A) Ponceau S stained nitrocellulose membrane (left panel), Western-blot (middle panel, anti-FLAG-HRP dilution at 1:800) and streptavidin-blot (right panel, streptavidin-HRP dilution at 1:10,000) of KAHRP-FLAG-APEX2 parasites treated and/or not treated with BP and H₂O₂. In all cases, material was generated by saponin lysis of iRBCs, which releases RBC cytosolic contents but retains parasite and exported membrane material, followed by solubilisation of all retained material using RIPA buffer. (B) NuPAGE of material enriched from the KAHRP-FLAG-APEX2 line following biotinylation, lysis with saponin and then RIPA, and purification of biotinylated proteins using streptavidin-coated beads enrichment. Eluted proteins stained by Coomassie-blue were only detected in the material from the BP+/H₂O₂+ condition (right panel). Legend for all gels/blots: first lane BP−/H₂O₂− (not treated with BP or H₂O₂); second lane BP−/H₂O₂+ (not treated with BP, treated with H₂O₂); third lane BP+/H₂O₂− (treated with BP but not treated with H₂O₂); fourth lane BP+/H₂O₂+ (treated with both BP and H₂O₂).