Figure 2. PfAtg8 is associated with membranes.

(A) Specificity of the two independently generated anti-PfAtg8 antibodies (#1 and #2). Crude antisera and purified antibodies were used for immunoblotting of lysates of asynchronized P. falciparum parasites. (B) Expression of PfAtg8 increases during the erythrocytic stage of development. Highly synchronized P. falciparum parasites were collected at 0, 12, 24, 32, and 40 h after invasion. The duration of one cycle of the erythrocyte stage was approximately 42 h. Expression levels of PfAtg8 were analyzed by immunoblotting. An antibody against HSP70 was used as a loading control. (C) PfAtg8 exogenously expressed in mammalian cells (lane 1), endogenous PfAtg8 expressed in P. falciparum (lane 2), and the mixture of these two samples were subjected to immunoblot analysis using anti-PfAtg8 antibody. (D) Lysates of asynchronized Plasmodium were separated into low-speed (13,000×g) pellet (LSP), high-speed (100,000×g) pellet (HSP), and high-speed supernatant (HSS) fractions, and analyzed by immunoblotting using anti-PfAtg8 antibody. (E) The LSP fraction prepared in (D) was treated with 2 M urea or 2% Triton-X 100 and separated into 100,000×g pellet (P) and supernatant (S). (F) Infected erythrocytes were cultured in the presence of the indicated concentration of chloroquine and expression of PfAtg8 was analyzed.