FIGURE 2.

Regulation of ErbB4 nuclear localization by PIAS3. A, COS-7 cells expressing HA-tagged ErbB4 JM-a CYT-2 with or without FLAG-tagged PIAS proteins were stained with anti-HA and photographed under a fluorescence microscope with a ×40 objective. B, expression of FLAG-tagged PIAS proteins in COS-7 cells was controlled by Western blotting with anti-FLAG. C, COS-7 cells expressing HA-tagged ErbB4 ICD2 and FLAG-tagged PIAS3 were subjected to subcellular fractionation. Nuclear and cytosolic fractions were analyzed by Western blotting (W) with indicated antibodies. Lamin B, nuclear marker; Mek1/2, cytosolic marker. D, COS-7 cells expressing HA-tagged ErbB4 JM-a CYT-2 and FLAG-tagged PIAS3 were treated for 4 h with 0 or 5 μm GSI IX and stained with anti-HA (red) and anti-PIAS3 (sc-46682, green). The nuclei were stained with DAPI (blue). The cells were visualized by confocal microscopy with a ×63 objective. E, COS-7 cells were transfected with constructs encoding HA-tagged ErbB4 JM-a CYT-2, ErbB4 JM-a CYT-2-V675A, or ErbB4 JM-a CYT-2-NLSI/II mutant and FLAG-tagged PIAS3 and stained with anti-HA. Cells were visualized by fluorescence microscopy and scored for cytosolic or nuclear staining. Columns (mean ± S.D.) show representative data from one of three independent experiments (*, p < 0.05). F, MCF-7 cells transfected with non-targeting (Qiagen) or PIAS3 siRNA were treated for 3 h with 25 ng/ml leptomycin B and for 45 min with 50 ng/ml NRG-1 and stained with anti-ErbB4 (HFR-1, red). The nuclei were stained with DAPI (blue). The cells were visualized by confocal microscopy with a ×40 objective. G, MCF-7 cells transfected with the indicated control siRNA (Ambion) or siRNAs targeting PIAS3 were subjected to subcellular fractionation. Nuclear and cytosolic fractions were analyzed by Western blotting with anti-ErbB4 (E200), anti-PIAS3 (sc-46682), anti-lamin B, and anti-Mek1/2. Loading was controlled by reblotting with anti-actin.