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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.

By Ling Yi (184648), Prabha Chandrasekaran (184653) and Sundararajan Venkatesan (184656)

Abstract

<p><b>A</b>) 10<sup>7</sup> 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>B</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. <b>C</b>) 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. <b>D</b>) 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.</p

Topics: Cell Biology, Molecular Biology, Physiology, Immunology, Developmental Biology, chemokine, lps, induced, rap-1, activation, disrupted, localization, activated, rap1, gtpase
Year: 2012
DOI identifier: 10.1371/journal.pone.0030404.g006
OAI identifier: oai:figshare.com:article/355105
Provided by: FigShare
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