EhARPC1 is recruited at the site of phagocytosis via EhAK1.

(A) and (B) Amoebic cells containing indicated constructs (EhAK1 or EhCaBP1) were grown for 48 h in the presence of 30μg/ml tetracycline (tet) and incubated with RBC for indicated times at 37°C. The cells were then fixed and immunostained with specific antibodies as indicated, and double stained with Alexa 488 (EhAK1/ EhCaBP1) or Pacific blue-410 (EhARPC1) labelled secondary antibodies. F-actin was stained with TRITC-phalloidin. Arrowheads indicate phagocytic cups formed in respective cell lines in absence of tetracycline and yellow color arrowheads indicate attached RBC in respective cell lines in presence of tetracycline. Alexa 488 staining of EhAK1/ EhCaBP1 in presence of tetracycline is pseudo-colored to gray. Scale Bar represents 10 μm. (C) and (D) Quantitative analysis of fluorescent signals of immunostained images of (A) is shown as a graph where N = 25 cells. One-way ANOVA test was used for statistical comparisons. (E) and (F) Western blot analysis of amoebic cells expressing indicated recombinant constructs showing the level of EhAK1 and EhARPC1 in tet-inducible vector alone, antisense EhAK1 (AS) or sense EhAK1(S) in the presence and the absence of tetracycline (30μg/ml). EhCaBP1 was used as an internal control. TOC is tet-o-CAT vector. “Two black star” p-value≤0.005.