Figure 3.

Depletion of cellular furin by exogenous α₁-PDX. Replicate plates of U-373 MG cells were incubated in the absence or presence of 8 μM α₁-PDX for 6 h. The cells were then either harvested directly (cont., control) or washed and incubated in fresh medium for an additional 10 h in the absence or presence of 50 μM cycloheximide (CHX) before harvesting. Crude membrane preparations from each sample were resolved by SDS/PAGE, and the levels of endogenous furin and transferrin receptor (TfR) were analyzed by Western blotting. The signals from the Western blot were quantified with National Institutes of Health image software. Black bars, furin; open bars, transferrin receptor; hatched bar, furin in the presence of cycloheximide. (Inset) U-373 MG cells were incubated with or without 8 μM α₁-PDX for 30 min. Membrane preparations from washed cells were resolved by SDS/PAGE, and furin molecules were identified by Western blotting by using antiserum PA1-062. Formation of the kinetically trapped SDS-stable complex between furin and α₁-PDX (EI*) causes furin to migrate at 160 kDa.