Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease
Article Title: RNA Binding Protein, HuR, Regulates SCN5A Expression Through Stabilizing MEF2C transcription factor mRNA
Figure Lengend Snippet: Hu Antigen R (HuR) is associated with myocyte enhancer factor‐2C ( MEF 2C) and protects MEF 2C mRNA from decay. A, HuR was associated with MEF 2C 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 MEF 2C 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 MEF 2C mRNA compared with the mRNA of MEF 2C measured immediately before addition of ActD. The MEF 2C 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 MEF 2C mRNA in cardiomyocytes with or without HuR overexpression was calculated by comparing the MEF 2C mRNA abundance at the given time point after blocking de novo transcription to the initial MEF 2C mRNA level. The percentages of remaining MEF 2C 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
Article Snippet: Real‐Time PCR Quantification Total RNA from cells was extracted using the RNeasy Mini plus Kit (Qiagen), according to the manufacturer's instruction, and 1 μg of total RNA was reverse transcribed into cDNA using SuperScript VILO Master Mix (Thermo Fisher Scientific, Waltham, MA), following the manufacturer's protocol.
Techniques: Isolation, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Staining, Agarose Gel Electrophoresis, Over Expression, Quantitative RT-PCR, Blocking Assay, Western Blot