Fig. S1. Detection of PfTRAMP in different stages by Western blotting using anti-PfTRAMP antisera raised in rabbits. Anti-PfTRAMP rabbit sera were used to probe parasite lysates made from different blood stages, namely, rings (R), trophozoites (T) and schizonts (S) by Western blotting. PfTRAMP is detected in all blood stages.


Fig. S2. Colocalization of PfTRAMP with endoplasmic reticulum marker, BiP, and Golgi marker, GRASP. In early trophozoites, PfTRAMP shows partial colocalization with both ER (BiP) and Golgi (GRASP) markers. The scale bar represents 2 μm.


Fig. S3. Localization of PfTRAMP-GFP in different asexual stages of transgenic parasites.

A. Fluorescent images of live transgenic parasites expressing PfTRAMP-GFP showing localization of the GFP fusion protein in different parasite stages. The scale bar represents 2 μm.

B. Detection of PfTRAMP-GFP and PfTRAMP in transgenic line at trophozoites and schizonts stages using anti-GFP mouse sera and anti-PfTRAMP rabbit sera.


Fig. S4. Detection of GFP in parasite lysates from wild type (WT) and transgenic P. falciparum schizonts expressing PfTRAMP-GFP by Western blotting. A band of ∼ 75 kDa, corresponding to PfTRAMP-GFP is recognized by anti-GFP specific sera in the transgenic, but not in the wild type (WT) parasite.


Fig. S5. Detection of proteins secreted from P. falciparum merozoites by Western blotting. P. falciparum merozoites were isolated in IC buffer followed by incubation in IC buffer, EC buffer and EC buffer containing erythrocyte membranes (EC + M). PfTRAMP is secreted when merozoites are resuspended in EC + M. PfAMA1 is secreted in EC buffer. There is no further secretion of AMA1 when merozoites are transferred to EC + M buffer. Merozoite pellets from each condition were probed with antisera against cytoplasmic protein NapL by Western blotting to control for number of merozoites used in each condition. Merozoite supernatants were also probed with antisera against NapL to control for merozoite lysis.


Fig. S6. Erythrocyte binding assays (EBAs) with native PfTRAMP. The full-length blot of the EBAs performed with native PfTRAMP using untreated (Un), neuraminidase (Neu), chymotrypsin (Chy) and trypsin (Try) treated erythrocytes. A single band of ∼ 28 kDa was detected using anti-PfTRAMP mouse sera.


Fig. S7. Erythrocyte binding assays (EBAs) performed using native and recombinant AMA1. No binding is detected with either native or recombinant AMA1 in EBAs. Preload (PL) indicates the native and recombinant AMA1used for EBA.


Fig. S8. Purified IgGs from rabbit sera raised against rPfTRAMP and rPfF2. Coomassie stained gel of purified IgGs from pre-immune rabbit sera (PIS), anti-PfF2 rabbit sera and anti-PfTRAMP rabbit sera separated under reducing (R) and non-reducing (NR) conditions. M, molecular weight markers.


Table S1. MSP typing of different field isolates and their origin.

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