Membrane Fractionation and Translocon Protein Detection

Membrane isolation and detection of translocon proteins was performed as described previously^{37 (link)}. Briefly, Vim^+/+ and Vim^−/− MEFs, seeded at 6×10⁶ cells per 10cm tissue culture-treated dish, were infected with early exponential phase bacteria in 2.5mL of DMEM supplemented with 10% FBS at an MOI of 100 for 1 hour at 37°C. For strains carrying an arabinose inducible construct, DMEM without glucose was used. The cells were washed 3 times with ice-cold PBS, scraped off the dish into ice-cold PBS containing protease inhibitors, and recovered by centrifugation at 225xg for 5 min at 25°C. The cell pellet was cooled on ice for 5 min and was resuspended in ice-cold 50mM Tris, pH 7.5 containing protease inhibitors and 0.2% saponin. After incubation on ice for 20 min and centrifugation at 16,100xg at 4°C for 30 min, the saponin-solubilized supernatant (cytosolic fraction) was collected, and the pellet was resuspended in 50mM Tris, pH 7.5 containing protease inhibitors and 1% Triton X-100. After additional incubation on ice for 30 min and centrifugation at 16,100xg at 4°C for 15 min, the Triton X-100-soluble supernatant (membrane fraction) was collected, and the pellet (detergent insoluble cellular debris and intact bacteria) was resuspended in 50mM Tris, pH 7.5 containing protease inhibitors and 1% Triton X-100. Western blots were performed using mouse anti-DnaK (Stressgen, SPA-880), rabbit anti-vimentin (Cell Signaling, 3932S), mouse anti-GAPDH (abcam, ab9484), rabbit anti-caveolin-1 (Sigma, C4490), rabbit anti-IpaC (gift from W. Picking), and rabbit anti-IpaB (gift of W. Picking).