Illustra Microspin G-25 columns (GE Healthcare) were used to remove unincorporated 32P. The primer Lorlatinib nmr extension reaction was performed using SuperScript III First-Strand Synthesis SuperMix (Invitrogen) following the supplied protocol. After first-strand synthesis RNA was degraded by incubation with RNase A (New England Biolabs) at 37°C for 15 min. Nucleic acids were precipitated by the

addition of 300 μl of chilled ethanol, incubation in a dry ice bath for 15 min, and centrifugation at 4°C. Dried samples were dissolved in loading buffer (98% deionized formamide, 10 mM EDTA, 0.025% xylene cyanol FF, 0.025% bromophenol blue) prior to loading on sequencing gel. Sequencing reactions were set up for each labeled primer using the SequiTherm EXCEL II DNA Sequencing Kit (Epicentre Technologies, Madison, WI). A PCR fragment amplified with the primers 145R7 and 146R1 was used as a template. Sequencing and primer extension reactions were loaded onto an 8% sequencing gel. After electrophoresis, the gel was dried and exposed to film at CHIR98014 price -80°C. NMR spectroscopy Strains 2019 wild-type and the

2019ΔcyaA ΔnagB mutant were grown in 100 ml cultures of sRPMI without Neu5Ac to early exponential phase. Neu5Ac, cAMP, or both were added and cultures were incubated for 20 min. Cells were pelleted and resuspended in 0.5 ml of MOPS buffer (40 mM MOPS, pH 7.3, with 50 μl D2O). Phosphorus NMR spectra were acquired at 162 MHz on a 400 MHz Varian Inova spectrometer in a 5 mm probe. Spectra were obtained upon excitation with at 45° pulse and digitization of 0.8 s followed by a delay of 1.7 s for

recovery between scans. Spectra 20 kHz wide were collected and processed with gaussian line-broadening of 0.1 s prior to Fourier transformation. Samples were maintained at 15°C, 2048 transients were averaged in an experiment lasting 1.5 hours. For each sample, two such spectra were collected one after the other. These were not significantly different, indicating that relatively minor changes take place on the time scale of data collection. However a third spectrum collected some 13 hours later indicated significant change in some cases. Chemical shifts were referenced relative to external 85% phosphoric acid at 0 ppm. Acknowledgements This work was supported by ACY-1215 supplier funding from NIAID Grants AI024616 and AI30040 and NIH grant GM085302. References ZD1839 datasheet 1. Greiner LL, Watanabe H, Phillips NJ, Shao J, Morgan A, Zaleski A, Gibson BW, Apicella MA: Nontypeable Haemophilus influenzae strain 2019 produces a biofilm containing N -acetylneuraminic acid that may mimic sialylated O-linked glycans. Infect Immun 2004,72(7):4249–4260.PubMedCrossRef 2. Mandrell RE, McLaughlin R, Aba Kwaik Y, Lesse A, Yamasaki R, Gibson B, Spinola SM, Apicella MA: Lipooligosaccharides (LOS) of some Haemophilus species mimic human glycosphingolipids, and some LOS are sialylated. Infect Immun 1992,60(4):1322–1328.PubMed 3.