Fig. 3.

HDAC inhibitors increase MSK1 protein levels in P19 cells. (A) Gel image: P19 cells were treated with 25 ng/ml TSA or 5 mM NaB for 0–4 hours as indicated and total cell lysates were prepared. Immunoblotting was performed to determine the expression levels of phospho-MSK1 and MSK1. Each blot was then reprobed with anti-β-actin as control. Graph: The pixel density values for phospho-MSK1, MSK1 and β-actin were measured for each time-point, and the relative changes in the ratio of phospho-MSK1/MSK1 and MSK1/β-actin was presented (TSA, left graph; NaB, right graph; *P < 0.05, **P < 0.01, n = 4). (B) P19 cells were pretreated for 1 hour with various concentrations of H89 (0, 5 or 10 μM) followed by TSA (25 ng/ml) or NaB (5 mM) stimulation for 6 hours. Total RNA was prepared and analyzed by qRT-PCR to determine changes in MOR expression levels as described previously (*P < 0.05, **P < 0.01, n = 4–8). (C) Gel image: P19 cells were treated with 10 μM VPA for 0–4 hours as indicated and total cell lysates were analyzed by immunoblotting with phospho-MSK1, MSK1 and β-actin antibodies. Histogram: P19 cells were pretreated for 1 hour with various concentrations of H89 (0–10 μM) followed by VPA treatment of 6 hours. MOR mRNA levels were determined by qRT-PCR as described in Fig. 1. Error bars represent the range of standard errors, and asterisks represent statistically significant findings (*P < 0.05; **P < 0.01, n = 3).