Figure 4.

Covalent binding of biotinylated LMB to hCRM1 in vivo. HeLa cells were preincubated with 0.1% ethanol (− competitor), 100 nM LMB (LMB), or 100 nM inactive LMB analog (i-LMB) for 1 h and then treated with or without 10 nM biotinylated LMB for 2 h. After cells were lysed, the proteins bound to biotinylated LMB were isolated by using immobilized streptavidin and analyzed by electrophoresis in an SDS/5–20% polyacrylamide gradient gel followed by silver staining (Left). A single protein emerged by incubation with biotinylated LMB in the absence of LMB competitor or in the presence of inactive LMB (arrow). The proteins resolved in the polyacrylamide gel were transferred to two poly(vinylidene difluoride) membranes, one that was immunostained with a rabbit polyclonal antiserum raised against hCRM1 (Center) and another that was stained with a horseradish peroxidase-conjugated anti-biotin antibody (Right) by using a chemiluminescence system. Asterisks indicate intrinsic biotin-binding proteins such as carboxylases.