FIG. 3.

Immunoprecipitation analyses of integrin expression on HEK293 cells. HEK293 (A) and A549 (B) cells (10⁷ cells) were detached with 1 mM EDTA and then biotinylated with 1 mg of sulfo-N-hydroxysuccinimide-biotin. After being washed with phosphate-buffered saline four times, labeled cells were then lysed in 1 ml of lysis buffer containing 1% Nonidet P-40 and protease inhibitors. Lysates from biotinylated cells (equal to 10⁶ cells) were immunoprecipitated with 2 μg of anti-integrin antibodies followed by capture with protein A/G beads. The immunoprecipitates were then separated on 6% sodium dodecyl sulfate gels under reducing conditions. After transfer to a polyvinylidene difluoride membrane, the filter was probed with a horseradish peroxidase-conjugated anti-biotin antibody.