Cellular fibronectin

Equivalent amounts of whole cell lysate were separated by 10% SDS-PAGE, and then transferred to a 0.45 μm nitrocellulose membrane. Membranes were blocked with 5% skim milk, and incubated overnight at 4 °C with the following antibodies: E-cadherin (BD Biosciences, San Jose, CA), cytokeratin-18 and cellular fibronectin (both from Sigma-Aldrich, Oakville, ON, Canada), vimentin, phospho-ERK1/2 mitogen activated protein kinase (MAPK), total-ERK1/2 MAPK, phospho-p38 MAPK, total-p38 MAPK, SLUG, SNAIL, or phospho-SMAD 2/3 (all from Cell Signaling, Danvers, MA). Membranes were then stripped and re-probed for the housekeeping protein, β-tubulin (Sigma-Aldrich, Oakville, ON, Canada).

Ring stage parasite samples were allowed to mature to the trophozoite/schizont stages during in vitro culture for 16–24 h, then mature parasite forms were enriched by gelatin flotation. Parasite binding inhibition assay was performed using a parasite suspension at 2–20% parasitaemia and 0.5% haematocrit [29 (link)]. 20 µl of recombinant endothelial receptors (CD36, ICAM-1, PECAM-1, P-selectin, integrin α₃β₁, integrin α₅β₁, integrin α_vβ₃, JAM-B, from R&D Systems Minneapolis, MN, cellular fibronectin, and laminin from placenta from Sigma, St. Louis, MO) at a concentration of 10 µg/ml were immobilized by adsorption on Petri dishes. The parasite suspension was preincubated with plasma diluted 1:5 for 30 min at room temperature then allowed to bind to the immobilized receptor for 30 min at room temperature. Plates were washed three times with PBS to remove unbound cells. Bound cells were fixed in 0.5% glutaraldehyde for 10 min, stained with 10% Giemsa for 2 min and quantified by counting the number of parasites in 20 high-power fields. Level of inhibition was defined based on the level of IE binding in the presence of a pool of plasma samples collected from malaria-naïve adults in the USA (negative control) as follow (100 − (N_test/N_control) × 100)). A minimum IE count of 20 in the presence of naïve plasma was required for the results to be accepted.

In the initial screening assay, binding of the rBucl8-CL-Ct to different
extracellular matrix (ECM) ligands was assessed by ELISA [31 (link)]. Wells were coated overnight with 1 μg
of each ligand dissolved in bicarbonate buffer: collagen type I and IV (Sigma),
elastin (Sigma), fibrinogen (Enzyme Research), plasma fibronectin (Sigma),
cellular fibronectin (Sigma), laminin (Gibco), and vitronectin (Sigma). Next, 1
μg per well of rBucl8-CL-Ct in TBS, 1% BSA was added and incubated for two hours
at 37°C. Wells were washed with TBS and bound rBucl8-CL-Ct was detected with
anti-6His-tag mouse mAb (Proteintech) in TBS-1% BSA and a secondary goat
anti-mouse HRP-conjugated Ab (Jackson Immuno Research Laboratories Inc.);
immunoreactivity was detected with ABTS substrate and measured
spectrophotometrically at OD₄₁₅. Data represent the mean ±SE of three
independent experiments (n = 3), each performed in triplicate wells.
Concentration-dependent binding was assessed in a similar manner, however with
varying concentrations (0–10 μM) of rBucl8-CL-Ct.