avium or 2D6 mutant were fixed and permeabilized at 4 h after infection. Antibody against SP-D protein was used and a second antibody labeled with Texas red was used. find more The arrows point to the green bacteria and red protein. Figure 4 Quantification of the SP = D protein expression assay in 100 U937 cells. The numbers represent the mean ± SD of three experiments. To investigate check details whether the complemented

M. avium 2D6 mutant phagosomes showed similar protein expression as that of wild-type, we infected the cells with 2D6 complemented bacteria [11] for 4 h, with MAC 109 as a positive control. The vacuoles containing the complemented M. avium 2D6 mutant showed expression of SP-D protein (Fig. 5A-5C) similarly to vacuoles containing the wild-type bacterium (Fig. 5D and 5E), though the percentage of infected cells showing the protein expression was 15% less than in macrophages infected with the wild-type I-BET151 chemical structure bacterium. Quantification of expression is shown in Fig. 4. Figure 5 Fluorescent microscopy images of U937 macrophages infected with fluorescein-labeled complemented M. avium 2D6 mutant. The SP-D protein is shown in red. Arrows point to bacteria (green) and SP-D protein (red). SP-D is present in macrophages infected with the MAC 104 strain and absent in the 2D6 mutant-infected macrophages. T-type

Ca++ channel is an integral membrane protein, which controls the rapid entry of Ca++ into excitable cells, and is activated by CaM-Kinase II (Swiss-Prot database). To verify our initial observation by MS/MS, we carried out parallel infection assays with fluorescein-labeled 2D6 and MAC 109 bacteria for 24 h. As shown in Fig. 6A and 6B, the majority of the cells infected with 2D6 mutant showed T-type Ca++ channel protein staining; whereas,

those infected with the wild-type MAC 109 and uninfected control U937 cells failed to express the protein (Fig. 6C and 6D, Fig. 6E and 6F, respectively). The observation was in agreement with the proteomic data showing that T-type Ca++ channel is expressed in mononuclear phagocytes infected with 2D6 attenuated mutant, but not when infected with MAC 109. Figure 6 Fluorescent microscopy Cediranib (AZD2171) images of U937 macrophages infected with fluorescein-labeled M. avium MAC 109 strain or 2D6 mutant. Macrophages were fixed and permeabilized 24 h after infection. Antibody anti-T-type Ca++ channel protein was used for 1 h, washed, and second antibody labeled with Texas red was applied for an additional hour. The arrows point to the green bacteria and red protein (A-F). To determine whether the phagosomes of macrophages infected with the complemented M. avium 2D6 mutant phagosomes failed to express the T-type Ca++ channel, mononuclear cells infected with complemented M. avium 2D6 bacteria and 2D6-attenuated mutant were evaluated. As shown in Fig. 7A and 7B, vacuoles with the complemented bacteria, in contrast to the 2D6 mutant (Fig. 7C and 7D), did not express T-type Ca++ channel protein.