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. 2017 Feb 7;292(13):5144–5165. doi: 10.1074/jbc.M116.743047

FIGURE 2.

FIGURE 2.

Mannosidase-II is recruited at the site of phagocytosis. A, RAW264.7 macrophages expressing mCherry-MAN2A (red) were incubated with mouse serum-coated latex beads for 30 min and 1 h. The images shown are representative of the 30-min samples. Right, total intensity of mCherry-MAN2A puncta on the bead surface was estimated using 3D spot creation module in Imaris 7.2, and the intensity distribution of the population has been plotted (see “Materials and Methods” for detail). The box plot represents data from more than 200 beads analyzed from two different experiments (values ± S.D.; scale bar, 4 μm). B, THP-1-derived macrophages were infected with GFP expressing E. coli (green, top panel) or S. typhimurium (green, lower panel) for 15 and 30 min. Cells were then stained with anti-mannosidase-II antibody followed by Alexa Fluor 568-tagged secondary antibody (red). Images shown are representative of the 15-min samples from both experiments. The bar plots at right show % co-localization of E. coli or Salmonella with mannosidase-II at the surface at 5 and 10 or 15 and 30 min, respectively, post-infection. Data represent the average of more than 200 bacteria from two different experiments (values ± S.D.; scale bar, 3 μm). C, mCherry-MAN2A (red)-expressing THP-1-derived macrophages were infected with PKH67-labeled H37Rv (green) for 1 and 2 h. Samples were fixed and visualized under the microscope. The bar plot at right shows % co-localization of H37Rv with mannosidase-II at both these time points. Data represent average of more than 200 bacteria from two different experiments (values ± S.D.; scale bar, 4 μm). D, THP-1-derived macrophages were infected with PKH67-labeled H37RV and fixed at the respective time points. These cells were then permeabilized using 0.05% Triton X-100 and stained with anti-mannosidase-II antibody followed by the secondary antibody tagged with Alexa Fluor 568, fixed, and visualized under the confocal microscope (scale bar, 3 μm). E, mouse BMDMs were infected with GFP expressing E. coli for 10 min, fixed, and stained with anti-MAN2A antibody (scale bar, 3 μm). DIC, differential interference contrast.