Journal: Infection and Immunity

Article Title: Identification of Acyloxyacyl Hydrolase, a Lipopolysaccharide- Detoxifying Enzyme, in the Murine Urinary Tract

doi: 10.1128/IAI.72.6.3171-3178.2004

Figure Lengend Snippet: Proximal tubule cells secrete pro-AOAH. (A) Diagram of AOAH structure, showing the conversion of the precursor (pro-AOAH) into mature AOAH. (B) Production of 35 S-AOAH by porcine proximal tubule cells in vitro. Labeled AOAH was immunoprecipitated as described in Methods and studied by SDS-PAGE and autoradiography. M, medium; L, lysate. Antibody +, anti-AOAH; antibody −, preimmune IgG. Only pro-AOAH is seen in the media, whereas the lysate contains both pro-AOAH and mature AOAH.

Article Snippet: Wells were blocked for 1 h at 37°C, samples and recombinant human AOAH standard were incubated at 4°C overnight or at 37°C for 2 h, secondary antibody (E553, purified polyclonal rabbit anti-human AOAH, final concentration 10 μg/ml) was added for 1 h at 37°C, biotin-conjugated goat anti-rabbit IgG (0.075 μg/ml) (Zymed, San Francisco, Calif.) was added for 1 h at 37°C, 2 μg of alkaline phosphatase-conjugated streptavidin (Jackson Laboratories)/ml was incubated for 30 min at 37°C, and the plate was developed with alkaline phosphatase substrate (5 mM Sigma 104 and 0.1 M Sigma alkaline buffer 221 in water).

Techniques: In Vitro, Labeling, Immunoprecipitation, SDS Page, Autoradiography