melitensis 16M that do not express mCherry After fixation, membr

melitensis 16M that do not express mCherry. After fixation, membrane permeabilisation with Triton X-100 (0.1% in dPBS) and AR-13324 chemical structure blocking of unspecific sites with bovine serum albumine (2% in dPBS), bacteria were detected with a monoclonal antibody raised against the lipopolysaccharides of Brucella (A76-12G12) [30] and a goat anti-mouse Texas Selleckchem GSK2118436 Red-conjugated secondary antibody. Fluorescence was observed using a Leica TCD confocal fluorescence

microscope. Western blotting MEFs were washed three times with PBS and then incubated for 10 min in cold lysis buffer (10 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.5% Triton X-100 and a protease-inhibitor cocktail (Roche)). After 10 min of rotation on a wheel, cell lysates were centrifuged for 15 min at 13,000 RPM at 4°C to sediment cell debris. Protein concentration of these clear lysates was determined using the BCA (Bicinchoninic acid) protein assay (Pierce). Fifteen micrograms

of proteins were separated by SDS-PAGE 12% and then, transferred onto polyvinyl difluoride (PVDF) membranes. Membranes were blocked for 1 h in PBS containing 0.1% Tween 20 and 2% of blocking agent (GE Healthcare), then incubated for 2 h with a primary monoclonal anti-LC3B antibody (NanoTools, Germany) and a secondary anti-mouse antibody conjugated to horseradish peroxidase (HRP). The activity of HRP was revealed by enhanced chemiluminescence BI-D1870 research buy (Perkin-Elmer). Statistical analysis Error bars indicate standard deviation (SD) or standard error of the mean (SEM) as indicated in the legend. Statistical significance was determined using SigmaPlot 11 software. Whenever possible, we have performed unpaired Student’s t-tests. When the normality test (Shapiro-Wilk) or the equal variance test failed, we carried out a Mann–Whitney rank sum test. A two-way ANOVA followed by a pairwise multiple comparison procedure (Holm-Sidak method) was also carried out. Statistical significant differences were accepted for p < 0.05. Ethics statement No live animal was used in this work. Acknowledgments We acknowledge Dr. Noboru Mizushima (Tokyo Medical and Dental University) for providing WT and Atg5−/− MEFs. This work was supported by the Actions de Recherches Concertées-Communauté Française

de Belgique (Grant number Convention N°08/13-015) and the University of Paclitaxel datasheet Namur. We thank Thierry Arnould and Martine Raes (URBC, University of Namur) for fruitful discussions and access to the confocal microscopy. Additional file Additional file 1: GFP-LC3 labelling in WT MEFs infected or not with B. abortus or B. melitensis. WT MEFs stably expressing GFP-LC3 were maintained under normal conditions (left) or under starved conditions (right). NI, BA and BM correspond to non infected cells, cells infected with B. abortus and cells infected with B. melitensis, respectively. MEFs were fixed at 10 h p.i. Bacteria were detected with a monoclonal anti-LPS antibody and an anti-mouse IgG Texas Red-conjugated secondary antibody. Nuclei were stained with DAPI.

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