Isolation of microbead and M.tb phagosomes. Six 15-cm plates of Raw264.7
Elesclomol were used for each condition. For isolation of the microbead phagosomal fraction, microbeads (2 μm, Polyscience) were added to Raw264.7 cells for 1 h, washed three times with prewarmed DMEM and incubated in DMEM with 10% FBS for the indicated times. Raw264.7 cells were then collected, lysed, and subjected to discontinuous sucrose gradient centrifugation as described previously [4]. For isolation of the M.tb phagosomal fraction, bacteria at an MOI of 10–30 were added to Raw264.7 cells in DMEM with 10% FBS for 1 h, washed and then incubated for the indicated times. Infected
cells were collected, lysed, and subjected to fractionation as described previously [15]. For immunoblotting analysis, aliquots of 12.5 μg of Raw264.7 cell lysate and 3 μg of phagosomal fraction proteins were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and then subjected to immunoblotting analysis using anti-Rab7 antibody (1:200 v/v), anti-LAMP-2 antibody (1:200 v/v), anti-cathepsin D antibody (1:200), and anti-cytochrome C (1:100 v/v). Band intensities from three independent experiments were quantified by Image J (http://rsb.info.nih.gov/ij/).
