Figure 3.

Association of guanosine triphosphate cyclohydrolase (GCH1) and Ecgp96 in human brain microvascular endothelial cells (HBMECs). A, Cytosolic fractions (300 µg) of uninfected HBMECs or HBMECs infected with OmpA+ Escherichia coli or Omp− E. coli were immunoprecipitated (IP) with anti-GCH1 antibodies. The immune complexes were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotted with anti-Ecgp96 antibodies. Similarly, total lysates were immunoprecipitated with anti-Ecgp96 antibody and immunoblotted with anti-GCH1 antibodies. Con, control. B, Plasma membrane fractions from total lysates of uninfected HBMECs or HBMECs infected with Omp+ E. coli or OmpA− E. coli were prepared. Membrane proteins (100 µg) were immunoprecipitated (IP) with anti-GCH1 antibodies and immunoblotted with antibodies to GCH1 or Ecgp96. The experiments were performed at least 3 times, and representative blots are shown here. The densities of protein bands from each blot were determined, normalized to the densities of immunoglobulin G (IgG), and graphed. The control (Cont) values were taken as one. Abbreviation: WB, Western blotting.