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. 2017 May 4;8(39):64840–64852. doi: 10.18632/oncotarget.17609

Figure 4. RASAL1 suppresses SRF activity via the PKA-LKB1-AMPK-MKL1 pathway.

Figure 4

a., AMPK inhibitors restored the suppression of SRF activity by RASAL1. SRF RE-luc was used to determine the suppressive effects of RASAL1 on SRF activity. AMPK inhibitors were added for 24 h before the assay. Data are means ± SD of three independent experiments in LX2 cells. *, p < 0.05 (t-test). b., Phosphorylation of MKL1 was increased by RASAL1 expression via AMPK activation. MKL1 proteins in LX2 control cells (control), RASAL1-overexpressing cells (RASAL1), and RASAL1-overexpressing cells after treatment with compound C for 6 h (RASAL1 + AMPK inhibitor) were immunoprecipitated. The phosphorylation status of MKL1 was determined by Western blotting using anti-phospho-serine antibodies. Immunoprecipitation using normal rabbit IgG was performed as a negative control. Representative results from three independent experiments are shown. c., Effect of RASAL1 expression on the phosphorylation status of AMPK. Control and RASAL1-expressing LX2 cells were treated with AMPK inhibitor for 1 h. Metformin, an activator of AMPK, was used as a positive control (p.c.) and was added for 1 h to control LX2 cells. Cell lysates were subjected to Western blotting using antibodies against the indicated proteins. Representative results from three independent experiments are shown. d., Phosphorylation of LKB1 and PKA was increased by RASAL1 expression. The phosphorylation and expression levels of the indicated proteins in control and RASAL1-expressing LX2 cells were determined. Representative results from three independent experiments are shown.