Fig 1: Characterization of STAT variant proteins used.A, Representative micrographs of HeLa cells expressing the indicated STAT variant proteins. The intracellular distributions of the following STAT-fluorophore fusion proteins are shown. Wild-type STAT1-6; N-domain mutated STATs (F77A, L78R/S, L82A); full-length STATs with heterologous signal sequence for either nuclear export (NES) or nuclear import (NLS) appended; full-length STAT1 with both the export and import signal sequences appended (NES-NLS); truncated STAT2 devoid of the C-terminus (ΔC); C-terminally truncated STAT2 with heterologous signal sequence for either nuclear export (NES) or nuclear import (NLS) appended. Presented are the green or red channels, and the Hoechst-stained cell nuclei (blue). Where there was treatment with leptomycin B (+LMB) it was for 4 h. The nuclei of interest are labelled with white asterisks to facilitate the matching of the GFP/Cherry signals and the Hoechst staining. B, Western blotting results using whole cell lysates (30 μl/lane) from HEK293T cells expressing mCherry fusion proteins of the full-length STATs or C-terminally truncated STAT2, as indicated. After blotting, total protein was stained with Ponceau S (bottom panel), followed by incubation with anti-mCherry antibody and immunodetection (top panel). In brief, experimental details were as follows. Twenty hours after transient transfection with vectors encoding STAT-mCherry fusion proteins as indicated, 4 × 106 HEK293T cells (transfection efficiency ∼70%) were lysed by boiling in 0.5 ml 2× SDS sample buffer (42 mM Tris-HCl, pH 6.8, 1.3% SDS (w/v), 233 mM DTT, 8.3% glycerol (v/v), 0.04 % Bromphenol blue). Proteins (30 μl/lane) were resolved by 7% SDS-PAGE followed by semi-dry protein transfer to a nitrocellulose membrane. Total protein loading was visualized by staining with 0.1% (w/v) Ponceau S in 1% (v/v) acetic acid for 1 min. Excess stain was removed with water (1 min), followed by blocking in 4% (w/v) dried skimmed milk powder in Tris-buffered saline containing 0.05% (v/v) Tween 20 (Sigma) and incubation with rabbit polyclonal anti-mCherry antibody (GTX128509, GeneTex). IRDye 800CW-coupled goat anti-rabbit secondary antibody (#926-32211) was from Li-Cor Bioscience. Blots were imaged by infrared fluorescence detection (Odyssey, Li-Cor Bioscience).