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. 2022 Sep 13;11:e80813. doi: 10.7554/eLife.80813

Figure 1. Generation of HA-tagged PfA-M17-HAglmS transgenic parasites.

(A) Schematic of the Pfa-m17 locus, and locus after single crossover recombination with pM17-HAglmS. The pM17-HAglmS plasmid contained the last kilobase of the coding sequence excluding the stop codon fused in frame to 3 x haemagglutinin (HA) and a single strep II (Str) tag. The plasmid also includes a glmS ribozyme with a synthetic untranslated region (UTR) and the selectable marker human dihydrofolate reductase (hDHFR). Arrows indicate oligonucleotides used in diagnostic PCRs as well as their expected sizes. (B) Diagnostic PCR showing integration of pM17-HAglmS at the endogenous locus. PCR was performed using the oligonucleotide pairs outlined in (A) on DNA extracted from parasites before (Pf3D7) or after (PfA-M17-HAglmS) transfection with the targeting construct. Oligonucleotides DO657 and DO658, which recognize the endogenous locus, serves as a positive control. (C) Western blot analysis of parasite lysates confirming HA expression. The predicted molecular mass of PfA-M17-HA is 72 kDa, and HSP70 serves as a loading control.

Figure 1—source data 1. Original gel electrophoresis presented in Figure 1B (panels 1 and 2).
Figure 1—source data 2. Marked up original gel electrophoresis presented in Figure 1B (panels 1 and 2).
Figure 1—source data 3. Original gel electrophoresis presented in Figure 1B (panel 3).
Figure 1—source data 4. Marked up original gel electrophoresis presented in Figure 1B.
Figure 1—source data 5. Original immunoblot presented in Figure 1C.
Figure 1—source data 6. Marked up original immunoblot presented in Figure 1C.

Figure 1.

Figure 1—figure supplement 1. Western blot of lysates prepared from mixed stage Pf3D7 wild type (WT) and PfA-M17-HAglmS parasites probed with either pre-bleed rabbit serum or rabbit serum after multiple rounds of inoculation with PfA-M17 recombinant protein (final bleed).

Figure 1—figure supplement 1.

The expected molecular mass of PfA-M17 is 68 kDa. Source data is provided in figure.
Figure 1—figure supplement 1—source data 1. Original immunoblot presented in Figure 1—figure supplement 1.
Figure 1—figure supplement 1—source data 2. Marked up original immunoblot presented in Figure 1—figure supplement 1.