Linearized plasmids were used as template for coupled transcription/translation. The addition of ε-labelled biotinylated lysine-tRNA complex to the reaction mixture allowed the biotinylation of the translated proteins.
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For each binding reaction, 1 pM biotinylated DNA template was coupled to 50-μg streptavidin-linked magnetic beads (Dynabeads M-280 Streptavidin; Invitrogen, Carlsbad, CA, USA).
The plasmids pEF6-Beclin-1-myc-His and pEF6-Beclin-1-D133A/D149A-myc-His were used as a template for in vitro coupled transcription/translation in a rabbit reticulocyte lysate system according to the manufacturer's instructions (Promega, Madison, WI, USA).
The following primer couples were used for generating in situ riboprobe templates: For Macpiwi, 5'-TGCTCAAGCTGGTGTTGCTGGTC-3' and 5'-GTCTTGTTGTTGTGCCGCGTGAG-3'.
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