Western Blot Analysis of O-Tagged VNAs and RNA-Encoded EfAb

Cell lysates and supernatants were loaded on 12% criterion TGX gels according to manufacturer’s instructions (Bio-Rad). Following SDS-PAGE, samples were transferred onto a 0.22 µm nitrocellulose membrane using the criterion blotting system (Bio-Rad). All washing and incubation steps have been described previously^{37 (link)}. For detection of O-tagged VNAs, the following antibodies have been used: OLLAS Epitope Tag Antibody (L2) from rat (Novusbio, 1:1,000) and a Rabbit anti-α/β tubulin (New England Biolabs, 1:1000) as primary antibodies, and Goat anti-Rat IgG (H + L) IRDye 800 CW (LI-COR, 1:15,000) as well as a Goat anti-Rabbit IgG (H + L) IRDye 680 RD (LI-COR, 1:15,000) as secondary antibodies. Primary antibodies were incubated for two hours. Secondary antibodies were incubated for one hour. For detection of RNA-encoded EfAb the following antibodies have been used: A Rabbit anti-tubulin UNLB (Cell Signaling Technology, 1:15,000) as primary antibody, a Goat anti-human IgG CW800 (LI-COR, 1:15,000) as well as a Goat anti-rabbit RD680 (LI-COR, 1:15,000) as secondary antibodies. Primary and secondary antibodies have been incubated for one hour. Protein detection and image processing were carried out in an Odyssey CLx^® Imaging system and Image Studio version 5.2.5 (LI-COR) according to manufacturer’s recommendations.