Figure 5.

Imp7 and Imp8 are required for TGF-β–activated Smad2/3 to translocate into the nucleus. (A) HeLa or HaCaT cells transfected with indicated siRNAs (40 nM) were analyzed by immunostaining using anti-Smad2/3 antibody, with or without prior TGF-β stimulation as indicated (100 pM, 30 min). The nuclei were marked by DAPI. Bars, 10 μm. (B) HaCaT cells transfected and treated as in A were examined by immunoblotting using antibodies recognizing phospho-Smad2 or total Smad2 and 3. (C) Total RNA was isolated from the same HaCaT cells as in B and the mRNA level of Smad7 was measured by quantitative real-time PCR. The plotted data are derived from three experiments and the error bars indicate SD. (D) HeLa cells were transfected with siRNA against Imp7 or Imp8 individually or in combination (20 nM each) and were stimulated with TGF-β before immunostaining as in A. Non-targeting control siRNA was used to balance the final concentration of total siRNA in each transfection (40 nM final). Bar, 10 μm. (E) Cells in D were categorized as nuclear (Smad2/3 predominantly in the nucleus) or cytoplasmic (Smad2/3 evenly distributed in the cytoplasm and nucleus) based on anti-Smad2/3 immunostaining pattern. Over 400 cells were counted in each case.