Figure 5.

Effect of various inhibitors of lumenal acidification of intracellular vesicles on the ability of FGF-2-CaaX to become prenylated in vivo. (A) NIH/3T3 cells were pretreated as in Figure 2A and then treated for 6 h with heparin and FGF-2-CaaX in the absence (lane 1) or presence of 1 μM monensin (lane 2), 10 nM bafilomycin A1 (lane 3), 20 mM NH₄Cl (lane 4), or 100 μM chloroquine (lane 5). After lysis, cellular material was analyzed as in Figure 2A. (B) NIH/3T3 cells were pretreated as in Figure 2A and then treated for 6 h with heparin and FGF-2-CaaX in the absence (lane 1) or the presence of increasing concentrations of concanamycin A (lanes 2-4). After lysis, cellular material was analyzed as in Figure 2A. (C) FGF-2 conjugated to Cy3 maleimide and FGF-2-CaaX conjugated to Alexa 488 maleimide were added to NIH/3T3 cells overexpressing FGFR1 and incubated for 2 h at 37°C. In some cases, the cells were pretreated with 10 nM bafilomycin A. The cells were then fixed and analyzed by confocal microscopy.