FIG. 2.
Arkadia acts downstream of TGF-β-induced Smad phosphorylation and nuclear accumulation. (A) HaCaT cells were transfected with the indicated siRNA SMARTpools, then treated with TGF-β (+ TGF-β) for 1 h or not treated with TGF-β (− TGF-β), and processed for immunofluorescence using an anti-Smad2/3 antibody and an anti-phosphorylated-Smad3 (anti-P-Smad3) antibody. Nuclei were visualized with 4′,6′-diamidino-2-phenylindole (DAPI). (B) HaCaT cells were transfected with the indicated siRNA SMARTpools and then treated with TGF-β or not treated with TGF-β for the indicated times. Whole-cell extracts were analyzed by Western blotting using antibodies against Smad3, P-Smad3, and against the Smad3 target genes PAI-1 and p21. Grb2 was used as a loading control. (C) Plots of Luciferase/Renilla values for HaCaT CAGA12-Luc/TK-Renilla cells transfected with the indicated siRNA SMARTpools and then treated with TGF-β or not treated with TGF-β.