Figure 3.

Effects of PAK on fDx uptake and efflux kinetics. (A) Effects of preincubation of cells with PDGF on the regulation of fDx uptake by PAK were examined. NIH3T3 cells expressing either vector alone (control), PAK1 wild type, or the indicated point mutants were maintained for 24 h in the absence of tetracycline to allow PAK1 protein expression. Cells were serum starved for 18 h during the expression period, and then PDGF (5 ng/ml) added to each cell line for 45 min. At the end of each incubation period, 5 mg/ml 70-kDa fDx was added and uptake measured at 0, 2.5, 5, 10, and 15 min. Cells were washed free of noninternalized fDx and the amount of fDx in cell lysates quantified by spectrofluorometry. The amount of fDx taken up per minute in the linear phase was plotted (±SE; n = 6). (B) NIH3T3 cell lines were handled as described above. After serum starvation for 18 h, PDGF and 70-kDa fDx were added to the cells for 45 min to allow uptake, and then washed three times in ice cold PBS and incubated in PBS containing 5.5 mM glucose, 1 mM CaCl₂, and 10 mM MgCl₂ for the indicated times. Cells were lysed and the fDx retained was quantitated by spectrofluorometry. One hundred percent represents the amount of fDx contained within each cell line at t = 0 min. SD for each data point was <18% of the mean (n = 6).