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This region of RNA is predicted to be predominantly unstructured in the context of these RNAs, agreeing well with the high level of modification we observe in our experiments.
We next evaluated the NB4 data set for possible detection bias, for example due either to differential efficiency of extracting distinct classes of protein in the lysates prepared, or to failure by MS to detect protein types featuring high levels of modification and/or unusual sequence combinations that are not cleaved efficiently into peptides.
AGE-specific fluorescence was detected at the excitation/emission wavelengths described elsewhere [ 22, 23] and were found to be very high for glycoaldehyde-treated BSA compared with native BSA, signifying the presence of a higher level of modifications.
The highest levels of modification of this protein occurs in the aged muscle.
Elucidation of the reason for this apparent increase in molecular weight warrants further investigations, but possible explanations include mutations (insertion) or a high level of post-translational modification.
However, in addition to the prevalence of H4R3me2s in the ventricular zone, high levels of this modification were detected in the post-mitotic neurons expressing NeuN marker within the cortical plate and subplate (Fig. 2A and B).
In summary, our screening of in vitro cell systems for the presence of immunochemically detectable levels of 5-hmC revealed a strong correlation of high levels of this modification with a pluripotent stem cell state.
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