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. 2014 Apr 17;10(4):e1004074. doi: 10.1371/journal.ppat.1004074

Figure 5. RNG2 is required for parasite growth.

Figure 5

(A) Schematic of the chromosomal locus of wild type RNG2 and the insertional mutant iΔHA-RNG2 showing the tetracycline regulatable promoter (t7s4), N-terminal HA tag, C-terminal cMyc tag (integrated separately) selectable marker DHFR, exon structure of RNG2 (black boxes), and primers (P1–3) used to verify correct integration. (B) PCR analysis of genomic rng2 locus after integration of the iΔHA insert. Primer P1–3 locations shown in (A) while P4,5 amplify tic22 as a positive control. (C) Western blot of three versions of tagged RNG2 using either HA or cMyc antibodies (equal protein loading in all lanes). (D) Western blot of HA-RNG2 expression after 0 to 3 days of anhydrotetracycline (ATc) treatment. Dense granule protein GRA8 used as a loading control. (E) Immuno-fluorescence detection of HA-RNG2 (green) and cell pellicles (IMC1, red) after no or one day of ATc treatment. Scale bar = 3 μm. (F) Plaque assay measuring parasite growth over 8 days of either the parental or knockdown cell lines, in the absence (−) or presence (+) of ATc.