2% serum at 37°C with shaking. Cultures were diluted 1:100 in fresh broth and allowed to shake at 37°C until they reached an absorbance of 1 at 600 nm (A600nm) corresponding to exponentially growing bacteria. For whole culture lysates

(NVP-HSP990 concentration samples labeled T, for total culture extracts as shown in Figures 2A and 3), cultures (6 ml) were incubated in the presence of lysostaphin (100 μg/ml) for 30 min at 37°C. To separate proteins in the culture medium (M) from those in the bacterial cell (C), cultures (6 ml) were centrifuged (10,000 ×  g for 10 min) and the supernatant was transferred to a new tube prior to lysostaphin treatment of intact cells. For subcellular localization of EssB (Figures 1A and 5 top panel), cultures NU7026 datasheet were centrifuged to separate medium and cells. Staphylococci were washed, and peptidoglycan digested with lysostaphin.

Staphylococcal extracts were subjected to ultracentrifugation at 100,000 ×  VX-661 clinical trial g for 40 min at 4°C. The supernatant, containing soluble proteins (S), was transferred to a new tube. The sediment containing insoluble membrane proteins (I), was suspended in 6 ml PBS buffer. Proteins in all samples were precipitated with 10% trichloroacetic acid on ice for 30 min. Precipitates were sedimented by centrifugation at 15,000 ×  g , washed, dried and solubilized in 100 μl of 0.5 M Tris–HCl (pH 8.0)/4% SDS and heated at 90°C for 10 min. Proteins were separated on SDS/PAGE and transferred to poly(vinylidene difluoride) membrane for immunoblot analysis with appropriate polyclonal antibodies. Immunoreactive signals were oxyclozanide revealed by using a secondary antibody coupled to IRDye© 680. Quantification of western blots

was conducted using a Li-Cor Biosciences Odyssey imager. Briefly, cells were grown to the same optical density. All strains reached similar density in the same time period suggesting that either deletion or cis -expression of genes did not affect growth of bacteria. Signal intensity of immune reactive signals for EsxA, EssB, EsaB and EsaD was compared to that obtained for WT, WT/vector, essB /p essB or WT/p essB sample extracts for Figures 2, 3, 5 A, B, C and D, respectively. Immune reactive signals (as shown in Figure 3) were averaged in three independent experiments and the data was analyzed in pairwise comparisons between WT/vector and variant strains with the unpaired two-tailed Student’s t -test and found to be statistically significant. Protein and polyclonal antibody purification Briefly, recombinant EssB, EssBNM, EssBMC, EssBΔM, tagged with N-terminal hexa-histidine were purified using Ni-NTA Agarose (Qiagen) following manufacturer’s recommendations.