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. 2014 Mar 28;12:22. doi: 10.1186/1478-811X-12-22

Figure 6.

Figure 6

JNK activation is required for TGFβ-induced ATF2 phosphorylation and CRP2 induction. (A) JNK activation is required for TGFβ-induced phosphorylation of ATF2 but not Smad2/3. VSMCs were pretreated with vehicle, JNK inhibitor SP600125 (10 μM), p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor U0126 (10 μM) for 30 min before stimulation with TGFβ (10 ng/ml). Phosphorylation of ATF2, JNK, p38, and ERK1/2 was determined 15 min after TGFβ stimulation. The membranes were subsequently probed with antibodies against total proteins for ATF2, JNK, p38, and ERK1/2 for loading control. (B) VSMCs were pretreated with vehicle or SP600125 for 30 min before stimulation with TGFβ. Cell lysates were harvested at the indicated time points and the phosphorylation of ATF2, JNK, and Smad2 examined. The membranes were subsequently probed with antibodies against total proteins for ATF2, JNK, and Smad2 for loading control. (C) JNK activity contributes to CRP2 induction. VSMCs were pretreated with vehicle or SP600125 for 30 min before stimulation with TGFβ for 24 h. Total proteins were then harvested for Western blot analysis to detect CRP2 expression. Values are mean ± S.E. of at least three experiments. *P < 0.05 vs. control (− TGFβ); #P < 0.05 vs. TGFβ-stimulated vehicle group. (D) Constitutively active FLAG-C2/ATF2 increases CRP2 expression. VSMCs were electroporated with control vector or FLAG-C2/ATF2 and total proteins prepared 24 h later. Western blot analysis was performed to assess CRP2 levels. Overexpression of FLAG-C2/ATF2 was evaluated by probing Western blots with FLAG antibody. (E) Overexpression of FLAG-C2/ATF2 rescues SP600125-inhibited TGFβ induction of CRP2. VSMCs were electroporated with control vector or FLAG-C2/ATF2, serum-starved, pretreated with vehicle or JNK inhibitor SP600125 for 30 min before stimulation with TGFβ for 24 h. Western blot analysis was performed to assess CRP2 expression. FLAG-C2/ATF2 expression was evaluated with FLAG antibody. (F) VSMCs were pretreated with vehicle or ROCK inhibitor Y-27632 (10 μM) for 30 min before stimulation with or without TGFβ for 10 min. Phospho-JNK expression was detected by Western blot analysis. The membranes were subsequently probed with actin for loading control. *P < 0.05 vs. control (− TGFβ); #P < 0.05 vs. TGFβ-stimulated vehicle group. Representative blots of at least three independent experiments are shown.