Adhesion Is Not Linked to clag9 Deletion was observed (data not shown) to human umbilical vein endothelial cells (HUVEC), a line to which some non-cytoadher- ent parasites are able to bind [46]. As several adhesins are implicated in the cytoadherence of PRBC, we further tested the binding of PRBC on purified CD36, ICAM-1, and some receptors rarely expressed in in vitro culture such as CSA and gC1qR. We did not observe any detectable binding of D10 to the tested receptors, which is in clear contrast to FCR3CD36 control adherence experiments (Fig. 1C and D). Repeated panning assays (3 times) on C32 cells and CD36 receptor did not yield any increase in adherence supporting the idea that D10 has irreversibly lost the adhesion phenotype. Characterization of the P. falciparum D10 variant PfEMP1 RT-PCR analysis using primers to amplify the conserved DBL1a domain indicated that a single type of var gene is transcribed by ring stage D10 parasites [47]. Northern blot analysis using a probe corresponding to the DBL1a domain of this var gene showed a unique major 8 kb transcript in ring stages (Fig. 2A, lane 1). This transcript was not observed in D10 trophozoites and schizonts (Fig. 2A, lane 2) or in any FCR3CD36 blood stages (data not shown). Successive hybridization with a conserved ATS probe confirmed that a single major transcript is present in ring stages of D10 (Fig. 2A, lanes 3, 4). Next, we verified whether the var transcript was translated into the protein PfEMP1 and expressed on the surface of the PRBC. Figure 2. Analysis of P. falciparum D10. A. Northern blot. Total RNA (10 mg) from P. falciparum D10 intra-erythrocytic stages hybridized with the D10 DBL1adomain of PfEMP1 probe (lanes 1–2), the expected 8 kb transcript was detected. The same membrane was hybridized with the ATS probe to control for loading (lanes 3–4). R: Ring stages (6–12 h after invasion), S: Schizonts (34–36 h after invasion). Size markers are in kb. B–D. Effect of trypsin on PfEMP1 of FCR3CD36 and D10 strains. B. Surface iodination. SDS-PAGE analysis of 125I-labelled FCR3CD36 (lanes 1–3) and D10 (lanes 4–6). C. Immunoprecipitation of FCR3CD36 (lanes 1–3) and D10 (lanes 4–6) TX100ins-SDSsol trypsin-treated extracts with a pool of human immune sera. Arrowheads indicate the PfEMP1 antigen. Molecular weight markers are in kDa. D. Western blot. Mature FCR3CD36 and D10 parasites were treated with 0 mg/ml (lanes 1 and 3), 10 mg/ml (lanes 2 and 4), or 100 mg/ml trypsin (lanes 3 and 6). TX100ins-SDSsol extracts were probed with goat anti-ATS and alkaline phosphatase-conjugated anti-goat and revealed with NBT/BCIP. The ATS antigen is shown by an arrow and an arrowhead indicates full- length PfEMP1. The same membrane was reacted with Hsp70 antibodies to control for loading. E. Surface immunofluorescence of FCR3CD36 and D10. Plasmion-selected parasites were treated with trypsin prior to incubation with pooled human immune sera (PIAG) followed by goat anti-human IgG Alexa Fluor 488. Scale bars: 5 mm. doi:10.1371/journal.pone.0029039.g002 PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e29039