FIG. 3.

Anisomycin, UV, and heat shock induce nuclear export of Net. (A) Cellular localization of GFP-Net and GFP-Net(L16A) exposed to stress conditions. NIH 3T3 cells transiently transfected with GFP-Net or GFP-Net(L16A) (see diagrams for structures) were treated with anisomycin (50 ng/ml), UV (40 J/m²), heat shock (42°C), sorbitol (600 mM), and TNF-α (20 ng/ml) and, as a control, were left untreated (Alone). After 30 min, cells were fixed and visualized by confocal microscopy. About 100 positive cells were analyzed to calculate the percentages with GFP-Net in the nucleus (N), cytoplasm (C), or both (B). (B) Nuclear exclusion of endogenous Net. NIH 3T3 cells were treated with anisomycin, UV, heat shock, TNF-α and sorbitol. The cells were fixed and stained with an antibody raised against Net (PAb 376), as shown for the anisomycin treatment. C, cytoplasmic; N, nuclear. Hoechst was used to stain nuclei. (C to E) Cellular localization of GFP-Net in the presence of cycloheximide and anisomycin. (C) Cells transfected with the GFP-Net expression vector were either left untreated (left) or pretreated with cycloheximide (CHX; 15 μg/ml) and then either mock treated (middle) or incubated with anisomycin (right). The middle panels show percentages of cells with GFP-Net in the nucleus (N), cytoplasm (C), and both (B). (D) Protein synthesis was measured by labelling with [³⁵S]methionine followed by SDS-PAGE and autoradiography. The cells were preincubated with [³⁵S]methionine for 15 min, and then at time zero (lane 1) incubation was continued in the absence (−) or presence (+) of cycloheximide for 2 or 4 h. (E) GFP-Net levels were determined by Western blotting of whole-cell extracts with anti-GFP antibodies before (lane 1) or after 2 h (lanes 2 and 3) or 4 h (lanes 4 and 5) of incubation in the absence (−) or presence (+) of cycloheximide. (F) Activation of endogenous p38 was monitored by Western blotting of whole-cell extracts with antibodies specific for dual-phosphorylated activated p38 (lanes 1 to 7) or ERK (lanes 8 to 11). The cells were either untreated (lanes 1 and 8), transfected with a vector expressing MKK3(Glu) (lane 3) or MKK6(Glu) (lane 5), treated with sorbitol (600 mM) after transfection with a vector expressing MKK3(Ala) (lane 4) or MKK6(Ala) (lane 6), treated with sorbitol (600 mM) without (lane 2) or with SB203580 (20 μM), transfected with a vector that expresses Ha-Ras (lane 9), and treated with EGF (0.1 μg/ml) in the absence (lane 10) or presence (lane 11) of PD98059 (20 μM).