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4) or 100�� (NA 1.45) oil immersion objectives. All image analysis was performed using Volocity 4.0.0 (Perkin Elmer). Cells were fixed in 2% (v/v) glutaraldehyde in 0.1?M Sorenson's phosphate buffer, http://www.selleckchem.com/products/GDC-0941.html pH 7.2, for 5?min before being scraped off of the coverslip and centrifuged. Fixation was continued at room temperature for at least a further 1?h. Cells were post-fixed in 1% (w/v) OsO4 in phosphate buffer at room temperature for 2?h, stained en bloc for 1?h with 1% (v/v) uranyl acetate in H2O, and then dehydrated and embedded in Epon resin. Eighty-nanometre-thick sections were collected on copper grids and stained with uranyl acetate and lead citrate. Sections were viewed using an FEI Tecnai 20 electron microscope, and images were captured using a Gatan Dualview camera. All experiments were performed at least in triplicate. For comparisons of means, paired t-tests or analysis of variance were used, as appropriate. A probability of http://en.wikipedia.org/wiki/Diglyceride grants from Cystic Fibrosis Canada (formerly the Canadian Cystic Fibrosis Foundation; CCFF) and the Canadian Institutes of Health Research (CIHR). R.S.F. is the recipient of a CIHR fellowship. V.J. is the recipient of a Heart and Stroke Foundation of Canada fellowship. K.K.H. was the recipient of CCFF and CIHR Graduate Studentships. M.A.V. holds a Canada Research Chair in Infectious Diseases and Microbial Pathogenesis. S.G. holds the Pitblado Chair in Cell Biology. Fig. S1. Immunoblot analysis of actin cross-linking. The ability of B. cenocepacia to induce covalent actin cross-linking was assessed by immunoblotting lysates of cells expressing both endogenous actin plus actin�CGFP, using anti-actin http://www.selleckchem.com/products/AZD0530.html (left) and anti-GFP antibodies (right). Note the difference in molecular weight between endogenous monomeric actin and the actin�CGFP fusion protein. Fig. S2. Effect of B. cenocepacia infection on actin in primary human macrophages. Primary human macrophages were left uninfected, exposed to dead bacteria, infected with live bacteria or treated with C. difficile toxin B. The cells were fixed, stained with phalloidin and imaged by spinning-disc microscopy. Representative XZ projections were constructed after serial optical sectioning. Bacteria are shown in red. Scale bars = 10 ��m. Fig. S3. Analysis of Rac1 by immunoblotting. Chemical modification and the integrity of Rac1 were analysed by immunoblotting using monoclonal antibodies. Lysates from cells that were exposed to dead bacteria, infected with live bacteria or treated with C. difficile toxin B, were probed using: (A) the anti-Rac antibody (clone 102); (B) anti-Rac antibody (clone 23A8) or (C) anti-GAPDH antibody, used for normalization of loading. The antibody produced by clone 102 does not recognize Rac when glucosylated by C.