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. 2020 May 29;9:e57799. doi: 10.7554/eLife.57799

Figure 2. P. falciparum cells express reporters with long polyA tracts.

(A) mRNA abundance of reporter constructs (+polyA36) by qRT-PCR relative to their counterpart lacking polyA stretches (-polyA36) in H. sapiens, T. thermophila, and P. falciparum cells. Data represent three biological replicates with a standard deviation. (B) Expression of reporter constructs in H. sapiens, T. thermophila, and P. falciparum followed by western blot analysis with αHA or αGFP antisera. Samples from two integrated clones for the -polyA36 control (-A) and the +polyA36 reporter (+A) are shown for T. thermophila. αβ-actin, α-Histone H3 trimethyl-lysine (H3kM) and αhDHFR are used as loading controls for western blot analysis from H. sapiens, T. thermophila and P. falciparum cells, respectively. (C) Images from live fluorescence microscopy of P. falciparum expression of reporter constructs with (+polyA36) and without (-polyA36) polyA tracts as well as parent (non-transfected) line, 2.5 µm scale bar. (D) Quantification of protein amounts for Thioredoxin-2HA-NanoLuciferase (Trx-2HA-NLuc) reporter without (-polyA36) and with 36 adenosine stretch (+polyA36) expressed in P. falciparum cells. Western blot analysis of Trx-2HA-NLuc reporter (Figure 2—figure supplement 2) and luminescence measurements were normalized to hDHFR or cell number, respectively. Luminescence data represent the mean value of three biological replicates with standard deviation.

Figure 2—source data 1. Luminescence and western blot quantification data.

Figure 2.

Figure 2—figure supplement 1. Generalized scheme of reporter constructs used for expression in H. sapiens, T. thermophila, and P. falciparum.

Figure 2—figure supplement 1.

Figure 2—figure supplement 2. Generalized schematic of Thioredoxin fusion NanoLuc reporter construct used for episomal expression in P. falciparum cells (Trx-2HA-Nluc).

Figure 2—figure supplement 2.

Lower: Western blot analysis of Trx-2HA-Nluc reporter expression in P. falciparum without (-polyA36) and with 36 adenosine stretch (+polyA36). Human DHFR (hDHFR) expressed from the same plasmid is used as loading and transfection control. Untransfected P. falciparum control cells (con) and Biorad Precision Plus Protein molecular weight markers (MWM) are indicated.
Figure 2—figure supplement 3. HA-pull-down assay of –polyA36, +polyA36 reporters.

Figure 2—figure supplement 3.

The input samples and the elution fractions were examined by western blot using anti-HA antibody HRP labeled (sc-7392HRP) anti-hDHFR (sc-377091). The HA-pull down was done using HA-magnetic beads (ThermoScientific, 88836) at 4°C over night. Bound proteins were eluted with sample buffer (BioRad).There is an equal amount of –polyA36, +polyA36 tagged reporter gene. The HA-tagged proteins were episomally expressed in HA-tagged pelota P. falciparum Dd2 strain.