Isolation of cytosol fraction from BMDMs

BMDMs were infected with the indicated E. coli strains or treated with Stx for 5 or 16 hours as indicated, and subcellular fractionation of BMDM was conducted by a digitonin-based fractionation method, as described previously (2). Briefly, the cells were washed and treated with 0.005% digitonin extraction buffer for 8 min to collect the supernatant containing cytosol. The residual cell fraction containing cell membrane, organelle, and nucleus was collected in 0.1% CHAPS buffer. Dilutions of these fractions were used for LAL assay to determine LPS quantity.