To support this finding, the surface location of ATP synthase β-subunit and β-actin on

HBMEC was demonstrated by immunofluorescence microscopy (Supporting Information, Fig. S1). These findings suggest that these proteins function as mannose-insensitive surface targets for FimH. To support this concept, we further characterized selleck kinase inhibitor the interaction between ATP synthase β-subunit and FimH. To verify the mannose-insensitive FimH binding to ATP synthase β-subunit of HBMEC, co-immunoprecipitation experiments of HBMEC lysates were performed in the presence of α-methyl mannose (100 mM). To minimize the nonspecific interaction with protein A agarose beads, the mixture of FimCH and HBMEC lysates were preincubated with protein A agarose beads, and the nonspecific complex was removed by centrifugation. The FimH–ATP synthase β-subunit complex was precipitated using anti-FimH antibody from HBMEC lysates preincubated with FimCH complex, as shown by Western blotting with anti-ATP synthase β-subunit antibody

(Fig. 2a). Controls for the nonspecific reaction of anti-FimH serum with ATP synthase β-subunit protein and rabbit serum (second and third lane of Fig. 2a, respectively) revealed see more no ATP synthase β-subunit co-immunoprecipitated from HBMEC lysates. We used the FimCH complex as a functionally active FimH, and then examined whether the FimC portion of the FimCH complex interacted with ATP synthase β-subunit by immunoprecipitating the mixture of biotinylated FimC and FimCH proteins and HBMEC lysate with

antibiotin antibody (Fig. 2b). Only ATP synthase β-subunit interacted with biotinylated FimCH (first lane), whereas crotamiton biotinylated FimC (second lane) and antibiotin antibody itself (third lane) did not reveal ATP synthase β-subunit from HBMEC lysates. For additional validation of the FimH interaction with ATP synthase β-subunit, we performed co-immunoprecipitation of HBMEC lysates and FimCH mixture with anti-ATP synthase β-subunit antibody, which was probed with anti-FimH antibody (Fig. 2c). FimH was detected only when anti-ATP synthase β-subunit antibody was used along with HBMEC lysates and FimCH (first lane of Fig. 2c). These lines of evidence indicate that ATP synthase β-subunit is the mannose-insensitive interacting target for FimH. We next examined whether anti-ATP synthase β-subunit antibody blocks the E. coli K1 binding to HBMEC in the presence of 10 mM α-methyl mannose. As shown in Table 3, anti-ATP synthase β-subunit antibody blocked the HBMEC binding of fim+ strain in a dose-dependent manner compared with anti-mouse IgG control, while it did not affect the binding of fim−E. coli to HBMEC (Table 3). However, 2 μg of anti-ATP synthase antibody did not decrease the HBMEC binding of fim+E. coli to the level of fim−E. coli (65% vs. 29% for fim+ and fim−E. coli, respectively).