Figure 2.

Stat1 contains a NES at position 302–314. Affinity purified fusions of GFP and GST linked to Stat1 helix 1 (a–c), helix 4 (d–f), the C terminus of helix 4 comprising amino acids 302–314 (g–l), and a Leu³⁰⁸Ala mutant of the NES (m–o) were comicroinjected with TRITC-labeled goat immunoglobulins into the nuclei of HeLa S3 cells. Cells treated with LMB starting 1 h before microinjection are shown in j–l. After 3 h incubation at 37°C, the cells were fixed and the nuclei stained with Hoechst 33258 dye. The intracellular distribution of the GFP fusion proteins harboring Stat1 fragments, the TRITC-conjugated injection control, and positions of nuclei are indicated in the fluorescence micrographs for each Stat1 derivative. Only fusion proteins containing the intact NES from helix 4 of the coiled-coil domain of Stat1 are transported into the cytoplasm. The presence of LMB (j) and the mutation of leucine³⁰⁸ (m) inhibit nucleocytoplasmic translocation.