Secretory Leukocyte Protease Inhibitor Binds to Annexin II, a Cofactor for Macrophage HIV-1 Infection

Ma, Ge; Greenwell-Wild, Teresa; Lei, Kejian; Jin, Wenwen; Swisher, Jennifer; Hardegen, Neil; Wild, Carl T.; Wahl, Sharon M.

Immunoprecipitation and Western Blot. 200-300 ϫ 10 6 monocytes in suspension or 7-10-d adhered macrophages were washed with PBS. Cell pellets were collected and incubated in sucrose lysis buffer (250 mM sucrose with complete EDTA-free protease inhibitor cocktail; Roche Applied Science) on ice, and then sonicated. The nuclear fractions were discarded after centrifugation (2,000 rpm at 4 Њ C) and the membrane fraction was resuspended in PBS with protease inhibitors, centrifuged (14,000 rpm for 30 min), and then the cytosol fraction was discarded. Membrane protein fractions were sonicated and proteins were quantitated (Bio-Rad Laboratories) before being precleared with normal IgG and UltraLink immobilized protein A/G beads (Pierce Chemical Co.) with rotation (30 min at 4 Њ C). The beads were pelleted (2,500 rpm) for 5 min at 4 Њ C. The supernatant was incubated with rhSLPI for 5-10 min on ice and rotated for 1-2 h at 4 Њ C with polyclonal goat anti-rhSLPI (Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-annexin II (IgG1; BD Biosciences), or anti-actin (IgG1; Santa Cruz Biotechnology, Inc.). UltraLink beads were added to this mixture and rotated overnight at 4 Њ C. The immunoprecipitates were boiled in reducing SDS-PAGE sample buffer after PBS wash and subjected to SDS-PAGE in Tris-glycine gels. For Western blot, the proteins were transferred to a nitrocellulose membrane and probed with 1 g/ml of polyclonal anti-SLPI (Santa Cruz Biotechnology, Inc.), anti-annexin II, anti-actin, or anti-␣ -tubulin (Sigma-Aldrich) followed by an appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.). Chemiluminescent signal was detected using the Supersignal West Pico system (Pierce Chemical Co.). Whole cell lysates from A431 or Jurkat cell lines were obtained from BD Transduction Laboratories.