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. 2016 Jan 15;48(1):e204. doi: 10.1038/emm.2015.98

Figure 3.

Figure 3

Isoproterenol increases HDAC6 by inhibiting c-Raf-MEK-ERK pathways. (a) Effects of MAPK inhibition on HDAC6 expression. H1299 cells were treated with 20 μM isoproterenol (ISO), 20 μM PD98059 (PD), 10 μM SP600215 (SP) or 20 μM SB03580 (SB) for 48 h, and HDAC6 expression was then analyzed by western blotting. (b) Effect of transcription inhibition on the MAPK inhibitor-induced increase in HDAC6 protein. H1299 cells were pretreated with 40 nM actinomycin D for 2 h and were then treated with 20 μM PD98059 (PD) for 46 h prior to western blotting analysis (filled bar). The HDAC6 mRNA level was assessed at 30 h by qPCR (empty bar). (c) The effects of isoproterenol on c-Raf-MEK-ERK signaling. H1299 cells were treated with isoproterenol for the indicated times, and the phosphorylations of c-Raf (filled bar), MEK (empty bar) and ERK (slant bar) were then analyzed by western blotting and densitometry. β-Actin was analyzed as a loading control. (d) Effects of ERK activation on the isoproterenol-induced increase in HDAC6 expression. H1299 cells were transfected with constitutively active MEK1 (caMEK1) for 24 h and were then treated with isoproterenol for 48 h prior to analysis. Filled bar represents HDAC6 expression and empty bar represents p-ERK. Asterisks (*) indicate significant differences from the respective control cells (P<0.05, Mann–Whitney U-test).