Figure 2. Expression, localisation and solubility profile of P. falciparum RhopH2.
(a) Western blot analysis of RhopH2-HA expression across the erythrocytic cycle. Immunoblots were probed with the antibodies as indicated. (b) Immunofluorescence analysis (IFA) on erythrocytes infected with PfRhopH2-HAglmS and fixed with acetone/methanol. RhopH2 is labeled with the anti-HA antibody. The bars represent 5 µm. (c) IFA on erythrocytes infected with PfRhopH2-HAglmS, fixed with acetone/methanol and probed with anti-HA (for RhopH2) and antibodies to the Maurer’s cleft protein SBP1 show that RhopH2 and SBP1 do not co-localise. (d) Solubility of RhopH2-HAglmS. Upper panel: Infected erythrocytes were synchronized and saponin-lysed when parasites reached ring (R) or schizont (S) stage and the pelleted material was sequentially dissolved in the buffers as indicated in the order of left to right (upper panel). Supernatant fractions were analysed by western blotting with the indicated antibodies. Insoluble material represents protein remaining in the pellet fraction after 1% Triton X-100 treatment. Lower panel: Alternatively, infected erythrocytes were saponin-lysed when parasites were at ring stages, split into equal portions and pelleted before dissolving in one of the indicated buffers. Both supernatant (Sn) and pellet (P) fractions were analysed by western blotting.