Mouse anti dnak

SPI1-induced cultures (10 mL) were prepared as detailed in the main Experimental Procedures. 1 mL of culture was pelleted by centrifugation (8,000 x g, 2 min, RT), supernatant removed and the pellet resuspended in 250 μL boiling 1.5x Laemmli SDS-PAGE sample buffer, boiled at 95°C for 10 min, and snap-frozen. In parallel, the remaining culture (~ 9 mL) was split between two pre-chilled thick-wall, polycarbonate ultra-centrifuge tubes (Beckman Coulter #355647) on ice and centrifuged at 30,000 x g in a MLA-80 rotor in an Optima Max Ultracentrifuge (Beckman Coulter; 20 min, 4°C). Supernatant was carefully removed, filtered through a 0.2 μm PES low-protein binding filter (GE Healthcare) and proteins were precipitated overnight at 4°C in 10% (v/v) trichloroacetic acid. Precipitated sample was divided into pre-chilled 2 mL snap-cap tubes and centrifuged for 20 min at 16,000 x g (4°C). Pellets were washed once with ice-cold acetone, centrifuged as above and allowed to dry for 5–10 min in a fume hood. Pellets were resuspended in a total volume of 200 μL pre-heated 1.5X Laemmli sample buffer (95°C), snap frozen and stored at -80°C until use. Aliquots of 10 μL were analyzed by SDS PAGE to determine secretion of relevant effector proteins. Antibodies used for protein detection were as follows: mouse anti-FLAG M2 (1:1000; Sigma-Aldrich) and mouse anti-DnaK (1:20,000; Enzo Lifesciences).