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Fig. 4. ARF6 involvement in actin polymerization. (A) ARF6 QS-EI inhibits bacterial entry. HeLa cells were transfected with the ARF6 QS-EI construct and infected for 4 hours with C. caviae GPIC on the following day. Extracellular and intracellular bacteria, as well as transfected cells, were labelled as described in Fig. 2D. The number of surface-associated and intracellular bacteria was counted in the transfected and non-transfected population (n>25 cells) and the efficiency of entry (intracellular/total cell-associated) was calculated. For each experiment, the efficiency of entry in transfected cells is expressed relative to that in non-transfected cells. Data are the mean±s.e.m. of three independent experiments. (B,C) ARF6 T27N and QS-EI mutants affect actin polymerization upon bacteria entry. Cells transfected or not with the ARF6 T27N-HA or the ARF6 QS-EI constructs were infected for 5 minutes as described in the Methods section. Actin was visualized with Texas Red-coupled phalloidin, ARF6 T27N-transfected cells were identified with anti-HA antibody and Alexa 488-labelled second antibody and cells transfected with the ARF6 QS-EI were visualized with anti-ARF6 antibody and Alexa 488-coupled second antibody. A representative experiment is shown in B. The arrows indicate actin patches. The actin patches visualized 5 minutes post-infection at the entry sites were quantified in transfected and non-transfected cells (C). Results are expressed as percent of patches in transfected cells compared with percent of patches in non-transfected cells. Data are the mean±s.e.m. of three independent experiments.
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