FIGURE 1.

IDE up-regulation in stress-exposed SHSY5Y, NHLF, and Jurkat cells. A, Western blotting analyses of IDE, HSP70, and GAPDH in lysates from heat-stressed SHSY5Y cells after 24 h of recovery. Three different stresses were used: heat stress for 15 and 30 min at 48 °C, oxidative stress using 1, 10, 50 μm H₂O₂ for 24 h, and nutrient deprivation for 60 min. A representative immunoblot of five independent experiments is shown. See supplemental Fig. S1 for the densitometric analysis. B, immunofluorescence analysis of heat-stressed SHSY5Y cells after 24 h of recovery: IDE content dramatically increases in the cytoplasm of SHSY5Y cells. Images were captured at different magnification (×20 and ×40). C, RT-PCR quantification of IDE mRNA in SHSY5Y-stressed cells: Heat stress for 30 min at 48 °C (white column); oxidative stress using 50 μm H₂O₂ (black column). IDE mRNA increases within 2 h of recovery and returns to basal levels within 5 h. Results are the means ± S.E. of five independent experiments and are displayed for treated cells as the relative expression of untreated controls. *, significantly different from control (p < 0.05, one-way ANOVA, followed by Tukey's test, n = 15). D, Western blotting analyses of IDE level in lysates from NHLF and Jurkat cells, respectively, incubated at 48 °C for 45 min and 60 min and at 42 °C for 30 min and 45 min, with increasing H₂O₂ concentrations ranging from 1 μm to 10 μm for 24 h and 60 min of serum starvation. See supplemental Fig. S3 for Western blotting analyses of IDE levels in lysates from PBL. The reported immunoblot represents five independent experiments. See supplemental Fig. S1 for the densitometric analysis. E, immunofluorescence analysis of heat-stressed NHLF cells after 24 h of recovery: the intracellular IDE content consistently increases. Images were captured at different magnifications (×20 and ×40).