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Fig. 2. F-actin and activated exogenous moesin polarize during early HIV-1 infection. (A) Western blot analysis of HIV-1 Env-induced phosphorylation of endogenous moesin in CEM cells overexpressing GFP fusions of full-length moesin (FL-moesin), the N-terminal FERM domain (N-moesin), or the C-terminal domain (C-moesin). A representative experiment is shown. ERM-P/ERM band density ratios from three independent experiments are shown beneath lanes. (B) ERM phosphorylation and subcellular localization of nucleofected moesin-GFP proteins in CEM cells either without infection (untreated, left panels) or 1 hour after HIV-1 infection (MOI, 1; right panels). Localization of exogenous moesin-GFP fusions was tracked by GFP fluorescence (GFP). Active ERM-P (Thr-P) was monitored with a specific Thr-P antibody and Alexa Fluor 568-labeled secondary antibody. The yz and xz planes are shown for each xy mid-section presented (arrows in the right panel). Data are from three independent experiments, presented as means ± s.e.m. Quantification of basal (untreated) and HIV-1 Env-mediated co-distribution of ERM-P and ERM is shown in parentheses; the percentages represent the number of cells showing co-distribution per 200 cells counted. (C) Distribution of F-actin and nucleofected moesin-GFP proteins in non-infected or HIV-1-infected (1 hour; MOI, 1) CEM cells. Quantification of HIV-1 Env-mediated co-distribution of endogenous moesin, nucleofected moesin-GFP constructs and F-actin are shown in parentheses. Scale bars: 10 µm. (D) Quantified flow cytometry of F-actin in non-infected CEM T cells that were either untransfected (Control) or nucleofected with GFP or FL-, N- or C-moesin-GFP. Data are from three independent experiments, presented as mean ± s.e.m.
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