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Whereas either chemokine or LPS treatment induced Rap-1 activation in monocytes, LPS treatment disrupted the leading edge localization of activated Rap1 GTPase and Epac-1, the Rap1 GEF.

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posted on 2012-02-09, 01:25 authored by Ling Yi, Prabha Chandrasekaran, Sundararajan Venkatesan

A) 107 monocytes were treated with or without LPS (2 ng/ml) for 15 min followed by 2 min treatment with 20 nM CCL2 or CXCL12. Cytoplasmic extracts were prepared and activated (GTP+) Rap1 was isolated using a commercial pull-down assay followed by SDS/PAGE and imunoblotting with anti-Rap1 mAb (upper). Cytoplasmic aliquots were analyzed directly for total cellular Rap1 (lower). B) Rap-1 levels were not significantly altered during a 15 min incubation with increasing amounts (2–100 ng/ml) of LPS. Cytoplasmic aliquots were analyzed directly for total cellular Rap1. The relative pixel values for Rap1 represent averaged results from using monocyte from three donors. C) LPS treatment abolished polarization of activated Rap1. Human monocytes on cover slips treated with or without LPS (2 ng/ml) for 15 min at 37°C were stimulated with 20 nM CCL2 for 2 min. Cells were fixed, permeabilized and incubated with Ral-GDS RBD GST (Thermo Scientific) for 30 min followed by staining with FITC conjugated anti-GST (BD FACS) and phalloidin-568. D) LPS treatment abolished polarization of Rap1 GEF, Epac-1. Monocytes on cover slips treated as above were fixed, permeabilized and incubated with anti-Epac-1 followed by staining with Alexa-488 conjugated anti-rabbit IgG and phalloidin-568. Individual channels corresponding to phalloidin and Rap1 and composite 2-color images are shown along with DIC images. Data are representative of 3 experiments with human monocytes from 3 different donors.

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