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. 2011 Jun 27;193(7):1305–1319. doi: 10.1083/jcb.201011143

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© 2011 Rivera and Brekken

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 6. — Endoglin associates with focal complexes in SPARC^−/− pericytes. (A) Endoglin colocalizes with vinculin plaques in SPARC^−/− pericytes. Cells were plated onto fibronectin-coated slides for 120 min. Cells were fixed and stained with antivinculin and antiendoglin IgG and visualized using a confocal microscope (TCS SP5; maximum intensity projections from 16–0.15-µm z stacks per stain were generated using ImageJ software and are presented). ZY planes are presented from the regions between the asterisks in split channel images. Bars, 20 µm. (B) FAK coprecipitates with endoglin immune complexes from SPARC^−/− pericytes. Endoglin was immunoprecipitated from lysates harvested from cells in suspension (S) or adhered to plastic (P) or fibronectin (F). Complexes were then probed for FAK or endoglin by SDS-PAGE and Western blotting. WCL, whole-cell lysates. (C) Recombinant SPARC reduces FAK-associated endoglin in SPARC^−/− pericytes. SPARC^−/− pericytes were incubated with either BSA or recombinant SPARC at 0, 50, or 150 µg/ml for 6 h. Endoglin was immunoprecipitated from cell lysates, and complexes were subjected to SDS-PAGE and Western blotting for endoglin and FAK. (D) Knockdown of endoglin reduces SMAD2 phosphorylation in SPARC^−/− pericytes. Pericytes were transfected with endoglin or control shRNA for 48 h. Lysates were prepared and subjected to SDS-PAGE. tSMAD2 and pSMAD2 were detected by Western blotting. pSMAD2 levels were normalized with ImageJ software. (E) Knockdown of endoglin reverses the effect of silencing SPARC on 10T1/2 cell transwell migration. Cells were transfected with the indicated shRNA for 48 h and then allowed to migrate in the indicated conditions. (F) Knockdown of endoglin reverses the effect of neutralizing SPARC on 10T1/2 cell transwell migration. Cells were transfected as in E and allowed to migrate in the presence or absence of 25 ng/ml anti-SPARC or control IgG as indicated. All experiments were performed at least twice with identical results. Mean values are presented. Error bars represent SEM (*, P < 0.05). WT, SPARC^+/+; KO, SPARC^−/−. Leica confocal images are presented in A (see Materials and methods). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; cntl, control; end, endoglin.