Figure 5 | Experimental & Molecular Medicine

Figure 5

From: Isoproterenol increases histone deacetylase 6 expression and cell migration by inhibiting ERK signaling via PKA and Epac pathways in human lung cancer cells

Figure 5

Isoproterenol inhibits c-Raf via the Epac-Rap1 pathways. (a) Effects of Epac knockdown on the 8-pCPT-cAMP-induced increase in HDAC6 expression. Epac1 and Epac2 or control siRNA were transfected for 24 h. Then 20 μM 8-pCPT-cAMP treatment was applied for 48 h prior to western blotting analysis. (b) Effects of isoproterenol on Rap1 activation. H1299 cells were treated with 20 μM ISO or 20 μM 8-pCPT-cAMP for 30 min. The GTP-bound Rap1 was then pulled down using the immobilized Rap1-binding domain (RBD) of RalGDS, and western blotting analysis was subsequently performed. (c) Effects of Rap1 activation on ERK phosphorylation (empty bar) and HDA6 expression (filled bar). H1299 cells were transfected with constitutively active Rap1A (caRap1A) and Rap1B (caRap1B) or EGFP control constructs (V) and incubated for 48 h prior to western blotting analysis. (d) Effects of Rap1A knockdown on 8-pCPT-cAMP-induced ERK phosphorylation and HDAC6 expression. H1299 cells were transfected with Rap1A shRNA, incubated for 24 h and then treated with 20 μM 8-pCPT-cAMP for 48 h. Asterisks (*) indicate significant differences from the respective control cells (P<0.05, Mann–Whitney U-test).

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