Transcend chemiluminescent non radioactive translation detection system

The in vitro translation analysis was performed using the TnT quick coupled transcription/translation system (Promega), following the manufacturer’s instructions. In brief, a T7 promoter was attached to the 5′-end of the three LETR1 transcripts and the luciferase ORF (positive control) by PCR amplification using Phusion high-fidelity DNA Polymerase (New England BioLabs). After gel purification, PCR fragments were used as templates to perform the in vitro translation reaction for 90 min at 30 °C. Transcend biotin-lysyl-tRNA (Promega) was used to label the newly synthesized proteins. Next, western blotting was performed largely as described above, with the exception of the usage of Bolt 12% Bis-Tris gels to be able to separate and keep small-sized proteins (micropeptides). Detection of biotinylated proteins was performed using the Transcend chemiluminescent non-radioactive translation detection system (Promega), following the manufacturer’s instructions. Blots were imaged on a ChemiDoc imaging system (Bio-Rad).

GST-E6 and GST proteins were expressed in Escherichia coli induced with IPTG (Sigma I-6758), and conjugated to Glutathione-Agarose beads (Sigma G-4510). The expression of GST-E6 and GST protein was confirmed by SDS-PAGE and Coomassie Blue staining. Myc, Myc mutants and Max proteins were translated in vitro with TNT^® T7 Quick Coupled Transcription/Translation System (Promega L1170). Myc fragments (Myc1-368, 143-368, 143-410, 143-439, 143-368+410-439, 1-368ΔMBI, 1-368ΔMBII, 1-368ΔMBI-II) were biotinylated during in vitro translation. The same amount of the IVT proteins were subjected to GST alone or GST-E6 pulldown assays. After electrophoresis with 4-20% of SDS-PAGE, the proteins were transferred to PVDF membranes and visualized with Transcend™ Chemiluminescent Non-Radioactive Translation Detection System (Promega, L5080) for Myc fragments and with blotting with Myc monoclaonal antibody (9E10) for wt Myc and its full-length based mutants (with deletions of either individual or combinations of MBI, MBII, HLH domains).