Fig. 2

ARF-6 is recruited to T. cruzi EA phagosome and its expression is required during host cell invasion. a: Wild-type MEF cells were treated with control siRNA, ARF-6 siRNA (Santa Cruz Biotechnology) as previously described (Silva et al., 2009). G-strain EAs were allowed to invade cells for space of one hour. Cells were then washed with PBS, fixed with Bouin and stained with Giemsa. The protocol was similar to the one described in Fig. 1. b: Wild-type MEF cell extracts were submitted to Sodium dodecyl sulfate- Polyacrylamide gel electrophoresis (SDS-PAGE) followed by electro-transfer into a nitrocellulose membrane for one hour at 250 mA per cm², incubation with polyclonal antibody anti-ARF-6 [diluted 1:100 in phosphate-buffered saline (PBS)] (Santa Cruz Biotechnology). After washes, membranes were incubated with peroxidase conjugated IgG anti-mouse (diluted 1:5000 in PBS) (Sigma-Aldrich) and developed by chemiluminescence (Silva et al., 2009). c: 5x10⁵ wild-type MEF cells/well were transfected with HA-ARF-6 plasmid (a gift from Prof. Dr. Philippe Chavrier, Department of Cell Biology, Research Center, Institut Curie.) and incubated with G-strain EAs for one hour. Cells were then formaldehyde-fixed and incubated with rabbit polyclonal antibody anti-HA (Santa Cruz Biotechnology) [diluted 1:100 in PBS + 0.02 % gelatin + 0.01 % azide (PGN)] followed by AlexaFluor® 488 conjugated IgG anti-rabbit (Invitrogen) (diluted 1:100 in PGN). Arrows indicate EA phagosome enriched in ARF-6. Bar: 10 μm. (p < 0.01)