Figure 2.

Inhibition of the proteasome has no effect on ligand-induced Met internalization but instead promotes recycling to the plasma membrane. (A) Cell-surface receptors were biotinylated on ice with a disulfide-cleavable biotin as described in MATERIALS AND METHODS. Cells were then rapidly rewarmed to 37°C and cultured in medium with or without HGF/SF and/or proteasome inhibitor for the indicated times to allow internalization of surface Met, after which they were rapidly cooled on ice, and remaining cell-surface biotin stripped with MESNA. After lysis, internalized biotinylated molecules were recovered with NeutrAvidin-conjugated beads, resolved by SDS-PAGE, and examined by Western analysis under reducing conditions using anti-Met^Hu intracellular-domain antibody. Similar results were obtained in three experiments. (B) To investigate recycling of Met after a 10-min HGF/SF “pulse,” cells were rewarmed after MESNA stripping, but in the absence of ligand, for 15 or 30 min at 37°C then subjected to a second round of MESNA stripping before lysis and analysis as described in A. Molecular mass markers are shown on the left.