Amplification, cloning and translation of ERG isoforms.

<p>A) Analysis of PCR-amplified <i>ERG</i> isoforms by electrophoresis on agarose gel. PCR products with length corresponding to ERG3 coding region (1458bp) were present in all samples. Calculated length of amplified coding region of predicted aberrant <i>ERG</i> isoforms was 775bp in NALM6 and ALL samples 1,2,4 and 5 (bearing the deletion of <i>ERG</i> exons 7–13) and 880bp in ALL sample 3 (bearing the deletion of <i>ERG</i> exons 7–11). PCR products corresponding to predicted aberrant <i>ERG</i> isoforms were present in all <i>ERG</i>del-positive samples and in none <i>ERG</i>del-negative samples (B-other cases: ALL-6 and ALL-7, hyperdiploid cases: ALL-8 and ALL-9, <i>ETV6/RUNX1</i>-positive case—ALL-10). (B) Schematic representation of <i>ERG</i> transcript variants cloned from NALM6 and of predicted encoded proteins. Regions encoded by alternative frames are displayed in grey. (C) Analysis of ERGaber proteins synthesized from PCR-prepared DNA templates by in vitro transcription/translation (T/T) assay. Proteins were detected by western blot using following antibodies: Anti-ERG antibody EPR3863 (Ab-N), Erg-1/2/3 Antibody C-17 (Ab-int), Erg-1/2/3 Antibody C-20 (Ab-C). In vitro T/T reaction without any DNA template served as a negative control (Neg. Ctrl.). ERGaberN was detected by Ab-N only and ERGaberC by Ab-C only. Ab-int showed only unspecific binding to proteins present in reticulocyte lysate used for T/T assay.