haemolyticus Dinaciclib has not been demonstrated. To investigate ChoP expression in H. haemolyticus, we obtained LOS profiles on silver-stained tricine SDS-PAGE from whole-cell lysates on three H. influenzae control strains, six H. haemolyticus strains containing a licA gene, and five H. haemolyticus strains lacking a licA gene [10]. As seen in Figure 1 (upper panel), both NT H. influenzae and H. haemolyticus demonstrated intra-and inter-strain variability in LOS migration. A duplicate gel was transferred to a Western

immunoblot and ChoP was detected with TEPC-15, a mAb that recognizes ChoP on a number of pathogenic bacteria [36–38]. TEPC-15 reacted with LOS-associated bands in all H. influenzae control strains and in Ferroptosis targets the six H. haemolyticus strains that contained a licA gene (Figure 1 lower panel). The antibody, however, did not react to five H. haemolyticus strains lacking a licA gene (Figure 1 lower panel). Figure 1 LOS profiles and TEPC-15 mAb reactivity in H. haemolyticus. H. influenzae and H. haemolyticus whole-cell lysates were run on tricine SDS-PAGE and silver stained to visualize LOS migration (upper panel) or transferred to nitrocellulose membrane for reactivity with the ChoP-specific mAb, TEPC-15 (lower panel). Lanes 1-3, H. influenzae ChoP phase-on variant strains

(E1a, Rd, and Mr15); lanes 4-9, H. haemolyticus strains hybridizing with a licA gene probe (M07-22, 60P3H1, 7P24 H, 3P41H5, C03-22, and H01-21); and lanes

10-14, H. haemolyticus strains not hybridizing with a licA gene probe (ATCC 33390, 3P18H1, 24P4 H, 26428, 26322) The association of ChoP epitopes with H. haemolyticus LOS was further supported by proteinase K digestion experiments. TEPC-15 reactivity was still present on Western immunoblots containing H. influenzae strain Rd and H. haemolyticus strain M07-22 that were pre-treated with proteinase K, although no proteins were visible in these preparations when they were run on glycine SDS-PAGE and stained with Coomassie (data not shown). Together these results suggest that, similar to H. influenzae, some strains of H. haemolyticus can express a ChoP epitope that is localized within its Endonuclease LOS. H. haemolyticus contains a lic1 locus similar to H. influenzae The ability of H. haemolyticus to hybridize with a H. influenzae licA gene probe suggests that H. haemolyticus contains a lic1 locus [10]. In H. haemolyticus strains M07-22 and 60P3H1, licA-licD gene probes were each found to hybridize with one restriction fragment on Southern blots, suggesting that all genes were confined to a single locus in each strain (data not shown). PCR designed to amplify overlapping regions of H. influenzae lic1 locus genes also amplified similar products in H.

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