Proteins present in only one run were not included. Immunofluorescence analysis Because some of the proteins identified in the phagosomes have not been previously described as part of the vacuole membrane, we attempted to confirm their presence by using immunofluorescence. Primary antibodies against pulmonary surfactant protein D (SP-D), T-type Ca++ alpha1I protein, EEA-1, CREB-1, MARCO and α-tubulin were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. Primary antibodies used were from rabbit, except

the goat anti-T-type Ca++ alpha1I. Secondary antibodies were Texas-Red conjugates (TR) and included donkey anti-rabbit IgG-TR (Amersham Biosciences, Piscataway, NJ) and mouse anti-goat IgG-TR (Santa Cruz Biotechnology, Santa Cruz, CA). The two-chamber slides from Nalge Nunc (Rochester, NY) were employed for macrophage monolayer preparation and fluorescence microscopy.

selleck The numbers of U937 cells were determined in a hemocytometer before seeding. A total of 5 × 105 cells were added in each tissue culture well of the two-chamber slides and were differentiated with 2 μg/ml of PMA overnight. The monolayers were then infected with MAC 109, 2D6 or the complemented 2D6 mutant labeled with NHS-CF as described above using a MOI of 10. The cells were incubated for 4 h at 37°C for SP-D protein expression and NCT-501 mouse 24 h for T-type Ca++ alpha1I protein expression. The time points were chosen based on the expression results. The chambers were washed three times with sterile phosphate buffer saline (PBS) and treated with 200 μg/ml amikacin to kill extracellular bacteria. The cells were subsequently washed and allowed to air dry. Cells were then fixed with 2% paraformaldehyde for 1 h at room temperature, permeabilized in cold 0.1% Triton X-100 (J.T. Baker) and 0.1% sodium citrate for 20 min on ice. Next, the monolayers were washed with PBS and blocked with 2% BSA (BSA, Sigma) in PBS for 20 min at room temperature. The 2% BSA was replaced with 1 ml of

specific primary antibody and allowed to incubate for 1 h. All the antibodies were prepared in 2% BSA in PBS to prevent non-specific binding. The cells were then washed three times with sterile PBS and re-incubated with the appropriate Texas-Red conjugated secondary antibody for an additional 1 h. Macrophages were washed three times with sterile PBS and allowed to air dry before Clomifene adding Aqua-mount mounting media (Lerner laboratories, Pittsburgh, PA) and cover slips (Corning, Corning, NY). Cell preparations were visualized with a Leica DMLB microscope. The microscope was operated by Spot 3rd Party Interface Software with a Photoshop CS version 8.0 on a Macintosh OS (version 4.0.9) based system. Immunoprecipitation and Western blot The U937 cells were infected with M. avium wild-type or 2D6 mutant with MOI 1 cell:100 selleck screening library bacteria in 75 mc2 flasks. After 30 min and 24 h following infections, monolayers were lysed and phagosomes were extracted as directed above.