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Conditioned medium (1.75?ml) from T?cells cultured in the absence or presence of S. Typhimurium was passed through a 0.22?��m filter. The resulting samples were aliquoted into 250?��l fractions that were precleared using 25?��l of protein A-coupled sepharose beads (Sigma), with agitation. After 1?hr of incubation at 4��C, the beads were pelleted by centrifugation and the supernatants were collected. Where noted, 22.5?��g of polyclonal rabbit anti-Escherichia coli L-Asparaginase II antibody or rabbit anti-mouse IgG control antibody Ceramidase (both Abcam) was added to the precleared supernatants. L-Asparaginase II of E.?coli is 96% identical to L-Asparaginase II of S. Typhimurium at the amino acid level, and T?cells treated with purified L-Asparaginase II of E.?coli (Sigma), like T?cells treated with purified L-Asparaginase II of S. Typhimurium, expressed levels of surface TCR-�� comparable to the levels expressed by T?cells cultured with WT S. Typhimurium ( Figure?S2G). After overnight incubation at 4��C with rotation, 25?��l of protein A-coupled sepharose beads were added to each sample. After an additional 3?hr of incubation Selleckchem Anticancer Compound Library at 4��C with agitation, the beads were pelleted by centrifugation and the resulting supernatants were used in T?cell assays. Statistical analysis of survival assays and organ burden assays was performed using the log-rank test and one-tailed, nonparametric Mann-Whitney test or two-way ANOVA, respectively. Statistical analysis of all other?assays was performed using a two-tailed paired t test (invasion assay)?or repeated-measures (randomized-block) one-way ANOVA with Bonferroni��s multiple-comparison post test, comparing selected pairs of columns. Statistical analysis was performed using Prism 5.0b (GraphPad Software), and p values?mTOR inhibitor grants awarded to A.V. (P01AI055621-Benach), H.A. (R21AI083964, R01AI083646 and R56AI077645), and M.M. (R01AI075093), as well as a USDA grant awarded to H.A. (AFRI CSREES 2009-03579) and?a?DOD grant awarded to M.M. (W81XWH-08-1-0720). A.V. was the recipient of a Harvard Digestive Diseases Center Pilot/Feasibility Award (P30DK034854-Lencer) and an Astellas Young Investigator Award (IDSA ERF/NFID). J.T. was the recipient of a Molecular and Cell Biology of Infectious Diseases Training Grant Fellowship (NIH T32AI007539-Bliska). A.T.