Figure 2.

msk, but not the importin β homologue ketel, is required for nuclear accumulation of endogenous Mad in Dpp-treated Drosophila S2 cells. (A) S2 cells were treated with indicated dsRNA and then subject to Dpp stimulation (1 nM for 1 h). Distribution of phospho-Mad (Mad-P) was detected by immunofluorescence staining using the PS1 antibody. The phospho-Mad signal per unit area in the nucleus and cytoplasm was measured using NIH ImageJ, and the nucleus/cytoplasm (N/C) ratios are shown (>50 cells were counted per sample). Bar, 10 μm. (B) S2 cells treated with indicated dsRNA were stimulated with Dpp as in A. Subcellular fractions were prepared and examined for phospho-Mad (Mad-P) and lamin levels (C: cytoplasm; N: nucleus). (C) S2R+ cells were subject to indicated RNAi. The cells were then stimulated with Dpp (1 nM) for 2 h and the mRNA level of dad was measured by real-time RT-PCR. The expression level of Rp49 was used as the internal standard for quantitation. The plotted data are derived from multiple experiments. Error bars indicate SD. (D) Co-immunoprecipitation of endogenous Msk with Flag-Mad. Whole-cell extract (WCE) was prepared from S2 cells transfected with Flag-Mad and Punt/Tkv as indicated and subject to immunoprecipitation using anti-Flag antibody conjugated to agarose beads. The bound proteins as well as input extract were analyzed by immunoblotting with indicated antibodies.