Figure 3.

Hu Antigen R (HuR) is associated with myocyte enhancer factor‐2C (MEF2C) and protects MEF2C mRNA from decay. A, HuR was associated with MEF2C mRNA. RNA was isolated and converted into cDNA using SuperScript VILO Master Mix after immunoprecipitation of RNA‐protein complexes from cardiomyocytes using either anti‐HuR antibody (Anti‐HuR) or control IgG. The reverse transcription–polymerase chain reaction (RT‐PCR) product was separated and visualized in ethidium bromide–stained agarose gel. B, Overexpression of HuR protected MEF2C mRNA from degradation. De novo transcription of the cells was blocked by addition of actinomycin D (ActD; 5 μg/mL) after 48 hours of doxycycline (Dox) induction. Total RNA was isolated from cells harvested at different time points after ActD addition. The abundance of mRNA was determined by real‐time quantitative RT‐PCR. The graph shows the percentage of the remaining MEF2C mRNA compared with the mRNA of MEF2C measured immediately before addition of ActD. The MEF2C mRNA was normalized by β‐actin mRNA measured at the same time point. The initial mRNA levels measured immediately before adding ActD were set to 100%. The percentage of remaining MEF2C mRNA in cardiomyocytes with or without HuR overexpression was calculated by comparing the MEF2C mRNA abundance at the given time point after blocking de novo transcription to the initial MEF2C mRNA level. The percentages of remaining MEF2C mRNA after transcription blocking are shown in the graph (n=4). C, Overexpressed HuR on Dox induction was confirmed by Western blot (WB) analysis using anti‐HuR antibody. *P<0.05.