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. 2010 Sep 8;285(46):35654–35664. doi: 10.1074/jbc.M110.128959

FIGURE 5.

FIGURE 5.

Smad-independent pathways influence Tgfβ induction of Arf. A, shown is a schematic diagram showing Smad-dependent and Smad-independent and specific inhibitors for each target: SB43, SB431542; SB20, SB203580; SP60, SP600125; U01, U0126; LY29, LY294002. B and C, shown is a representative Western blot for the indicated proteins from wild type MEFs (B) and β-galactosidase activity in ArflacZ/lacZ MEFs (C) exposed to Tgfβ2 (T2) or vehicle (V) for 48 h and either DMSO or the indicated chemical inhibitors. Quantitative analysis (C) shows that Tgfβ2 significantly changed β-galactosidase activity when compared with relevant vehicle (p < 0.013); however, the induction of β-galactosidase was significantly blunted by SB203580 and SB431542 when compared with DMSO (p = 0.0015 (#) and <0.001 (*), respectively), and it was significantly enhanced by U0126 when compared with DMSO (p = 0.047 (#)). D, a representative Western blot shows the corresponding targets for individual inhibitors in ArflacZ/lacZ MEFs exposed to Tgfβ2, and the indicated chemical inhibitor confirms that LY294002 blocks AKT phosphorylation (lane 4), U0126 blocks p42/44 phosphorylation (lane 6), and SB431542 blocks Smad2 and p38 MAPK phosphorylation (lane 7). E, representative Western blot using ArflacZ/lacZ MEFs exposed to the indicated chemical inhibitors or DMSO for 20 min shows that SB20 blunts anisomycin-stimulated, p38 MAPK-dependent phosphorylation ATF-2. F, a representative Western blot using ArflacZ/lacZ MEFs exposed SP600125 for 20 min blunts UV-stimulated phosphorylation of JNK.