Fig. 8. Bet3 acts before the EGTA-sensitive step in ER-Golgi transport in vitro. As outlined in (A), permeabilized NRK cells, cytosol and ATP were incubated in the absence (lanes 1 and 2) or presence of 8 mM EGTA (lanes 3-12) on ice (lane 1) or at 32°C (lanes 2-12) for 60 minutes (stage I). The membranes were then collected by a brief spin and resuspended in fresh transport cocktail including fresh ATP and cytosol (lanes 2-11) or Bet3 immunodepleted cytosol (Bet3 drlc, lane 12). Reagents were added at stage I or stage II as indicated in (C). Transport was measured by the conversion of the VSV-G protein to the endo-H-resistant form (B) and quantified (C).