To ensure proper phase separation, a known detergent phase protein

and a soluble aqueous phase protein, OspA and BB0796, respectively, were included as controls. B. BB0324 and BB0028 are localized to the B. burgdorferi OM. OM and PC fractions from B. burgdorferi B31-A3-LK cells were isolated as described in Methods. Whole-cell equivalents from each fraction were subjected to SDS-PAGE and immunoblot analysis using BB0324 or BB0028 antisera. For MK-4827 positive controls, fractions were immunoblotted with antibodies against BamA and the known OM lipoprotein Lp6.6, which is anchored to the inner leaflet of the B. burgdorferi OM. To verify OM purity, fractions were also immunoblotted with antibodies against the inner membrane lipoprotein OppAIV. C. BB0324 and BB0028 are subsurface selleck chemicals llc proteins. Whole-cell lysates of B. burgdorferi B31 cells were either mock-treated (-) or proteinase K-treated (+) before being immunoblotted with BB0324 or BB0028 antisera. As a positive control for PK activity, samples were probed with antibodies

to BB0405, a known surface-exposed OMP. The mock-treated and the PK-treated samples were also immunoblotted with GDC-0068 rabbit anti-FlaB antibodies to ensure equal loading. D. Subsurface BB0324 and BB0028 proteins are degraded Nintedanib (BIBF 1120) by proteinase K. B. burgdorferi cell membranes were disrupted with detergent and lysozyme prior to incubating the lysates in the absence

(-) or presence (+) of proteinase K. Samples were immunoblotted using antibodies to BB0324, BB0028, or FlaB (a known periplasmic protein). We next examined the cellular location of BB0324 and BB0028 to confirm their presence in the OM. As shown in Figure 6B, BB0324 and BB0028 were detected in the isolated OMs of B. burgdorferi, demonstrating that both proteins are localized to the OM. Cell fractions were also probed with antibodies to the OM-localized BamA and Lp6.6 proteins, as well as to the IM-anchored OppAIV lipoprotein, to verify OM specificity and purity. To determine if BB0324 or BB0028 are anchored to the periplasmic leaflet of the OM, we next incubated whole B. burgdorferi cells in the presence or absence of proteinase K (PK). These experiments revealed that there was no difference between mock- or PK-treated samples when probed with anti-BB0324 or anti-BB0028, indicating neither protein is surface-exposed (Figure 6C). As controls for PK activity and OM integrity, lysates from the mock- and PK-treated cells were also probed with antibodies against the surface-localized BB0405 protein [32, 39] and the periplasmic FlaB protein, respectively.